Methods and compositions useful for treating diseases involving Bcl-2 family proteins with isoquinoline and quinoline derivatives

ABSTRACT

The present invention relates to a compositions for and methods for cancer treatment, for example, hematopoietic cancers (e.g. B-cell Lymphoma). In other aspects, the invention provides methods for treating particular types of hematopoietic cancers, such as B-cell lymphoma, using a combination of one or more of the disclosed compounds and, for example, 26S proteasome inhibitors, such as, for example, Bortezomib. In another aspect the present invention relates to autoimmune treatment with the disclosed compounds. In another aspect, this invention relates to methods for identifying compounds, for example, compounds of the BH3 mimic class, that have unique in vitro properties that predict in vivo efficacy against B-cell lymphoma tumors and other cancers as well as autoimmune disease.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/646,586, having a 35 U.S.C. § 371(c) date of May 21, 2015,which is the National Stage of International Application No.PCT/US2013/071285, filed Nov. 21, 2013, which claims priority to U.S.Provisional Application No. 61/729,251, filed on Nov. 21, 2012, thecontents of each of which are herein incorporated by reference in theirentireties.

FIELD OF THE INVENTION

This invention relates generally to methods and compositions fortreating cancer and autoimmune diseases. Cancer may includehematological malignancies, such as Multiple Myeloma and B-celllymphoma.

BACKGROUND

Currently the prevalence of Multiple Myeloma is 63,000 people in theU.S. with about 13,000 new cases per year. There are 360,000 cases ofnon-Hodgkin's lymphoma (NHL) in the United States and 550,000 worldwide,with about 56,000 cases diagnosed per annum and 23,000 deaths per annum(See American Cancer Society, SEER CANCER STATISTICS REVIEW 1975-2009(Vintage 2009 Populations)). Twenty percent of these do not respond tocurrent therapy. In terms of all NHL cases, 60% are aggressive, of which50% do not respond to front line therapy. In addition, chroniclymphocytic leukemia (CLL) is the most common form of adult leukemia inthe U.S. and in most of Western Europe. There are approximately 70,000cases of CLL in the U.S., with 10,000 new cases diagnosed per annum (SeeNCI, What You Need To Know About: Leukemia booklet). CLL patients have apoor survival prognosis with a five-year survival rate of 46%.

Mcl-1 is a key regulator of lymphoid cancers including multiple myeloma(MM) (Zhang, et al. (2002), Blood 99:1885-1893), non-Hodgkin's lymphomas(Cho-Vega, et. al (2004) Hum. Pathol. 35(9): 1095-100) and chroniclymphocytic leukemia (CLL) (Michaels, et al. (2004), Oncogene 23:4818-4827). Additionally, treatment of myeloma cells with the proteasomeinhibitor Bortezomib (VELCADE) has been shown to cause elevated Mcl-1expression (Nencioni, et al. (2005), Blood 105(8): 3255-62) and this isseen in some myeloma patients (Podar, et al. (2008) Oncogene 27(6):721-31). It is proposed that an Mcl-1 inhibitor would enhance theefficacy of VELCADE treatment in MM patients.

Though Rituxan, which targets the B-cell surface protein CD-20, hasproven to be a valuable front line therapeutic for many NHL and CLLpatients, resistance to this drug has been shown to correlate withelevated expression of B-cell lymphoma 2 (Bcl-2) or Myeloid Cellfactor-1 (Mcl-1) proteins (Hanada, et al. (1993) Blood 82: 1820-28;Bannerji, et al. (2003) J. Clin. Oncol. 21(8): 1466-71). Notably, thereis a high correlation of elevated Mcl-1 with non-responsiveness tochemotherapies in B-cells from CLL patients. (Kitada, et al. (2002)Oncogene 21: 3459-74). Rituxan-resistant CLL cells also have a higherMcl-1/Bax ratio than normal cells, while there is no significantcorrelation of the Bcl-2/Bax ratio. (Bannerji, et al. (2003) supra).

Moreover, approximately 30% of diffuse large cell lymphomas (DLCLs) haveincreased Bcl-2 expression levels. This correlates with poor patientresponse to treatment with combination chemotherapy (Mounier, et al.(2003) Blood 101: 4279-84). In follicular non-Hodgkin's lymphomas andplasma cell myeloma, Mcl-1 expression positively correlates withincreasing grade of the disease (Cho-Vega, et al. (2004) Hum. Pathol.35(9): 1095-100).

The value of Bcl-2 as a target in anti-tumor therapy has been wellestablished. The literature also reports on Mcl-1 as a target intreating NHL, CLL, and acute mylogenous leukemia (AML) (Derenne, et al.(2002) Blood, 100: 194-99; Kitada, et al. (2004) J. Nat. Canc. Inst. 96:642-43; Petlickovski, et al. (3018) Blood 105: 4820-28). Researchershave recognized that proteins in the Bcl-2 family regulate apoptosis andare key effectors of tumorigenesis (Reed, (2002) Nat Rev. Drug Discov.1(2): 111-21). Bcl-2 promotes cell survival and normal cell growth andis expressed in many types of cells including lymphocytes, neurons and,self-renewing cells, such as basal epithelial cells and hematopoieticprogenitor cells in the bone marrow.

In many cancers, anti-apoptotic Bcl-2 proteins, such as Bcl-2 and Mcl-1,unfortunately block the sensitivity of tumor cells to cytostatic orapoptosis inducing drugs. These proteins are therefore targets foranti-tumor therapy. A recently described class of small molecules thatinhibit Bcl-2 family proteins are the BH3 mimetic compounds (Andersen,et al. (2005) Nat. Rev. Drug Discov. 4: 399-409). These compoundsfunction by inhibiting BH3 mediated protein/protein interactions amongthe Bcl-2 family proteins. Several studies have described BH3 mimeticsmall molecules that function as Bcl-2 inhibitors by blocking BH3binding (reviewed in Reed, et al. (2005) Blood 106: 408-418). Compoundswith BH3 mimic function include HA-14-1 (Wang, et al. (2000) Proc. Natl.Acad. Sci. USA 97: 7124-9), Antimycin-A (Tzung, et al. (2001) Nat. Cell.Biol. 3: 183-191), BH3I-1 and BH3I-2 (Degterev, et al. (2001) Nat. Cell.Biol. 3: 173-82), and seven un-named compounds (Enyedy, et al. (2001) J.Med Chem 44: 4313-24), as well as a series of terphenyl derivatives(Kutzki, et al. (2002) J. Am. Chem. Soc. 124: 11838-9), and two nowclasses of molecules (Rosenberg, et al. (2004) Anal. Biochem. 328:131-8). More recently, a BH3 mimic compound has been tested in a mousetumor model (Oltersdorf, et al. (2005) Nature 435: 677-81).

The promise for using BH3 mimetic compounds as anti-tumor therapeuticshas been recognized. However, to date there are no conclusive reportsfrom the clinic on the efficacy of any anti-cancer drug with this modeof action. While pharmacological manipulation of the Bcl-2 familyproteins is a feasible approach to achieving therapeutic benefit forcancer patients, the complexity of the network of proteins that comprisethis family makes this prospect difficult. Therefore, with the largeunmet medical need for treating hematological malignancies, newapproaches to assessing and utilizing the detailed activity of the BH3mimetic molecules will have value in developing this class oftherapeutics.

SUMMARY OF THE INVENTION

Accordingly, the present invention is based on the discovery of methodsand compounds useful for treating cancer and autoimmune diseases,including methods involving the manipulation of Bcl-2 family proteins.

The present invention relates generally to methods and compositions fortreating cancer and autoimmune diseases. Cancer may includehematological malignancies, such as Multiple Myeloma and B-celllymphoma. More specifically, the invention relates to treating cancersincluding hematological malignancies, with compounds that inhibit theBcl-2 family protein Mcl-1 as well as other of the Bcl-2 familyproteins. In addition, this invention relates to methods for determiningselectivity of newly classified “BH3 mimic” compounds to predictefficacy in treating hematological and other malignancies involvingMcl-1.

In one aspect, the invention pertains to methods of using compounds ofFormula I in the treatment of various disease states, including thoseinvolving Bcl-2 family proteins, where Formula I is:

or a stereoisomer thereof, tautomer thereof, solvate thereof, or apharmaceutically acceptable salt thereof, wherein:

X is selected from O, C, or N, with C or N optionally substituted withhydrogen, substituted or unsubstituted C₁₋₆ alkyl, hydroxyl-substitutedalkyl, substituted or unsubstituted C₅₋₁₀ aryl, or substituted carbonylgroup;

Y is CH or N;

Z is C or N;

Z′ is CH or N; and

when Z is C, R₁ is selected from hydrogen, halogen, substituted orunsubstituted C₁₋₆ alkyl, substituted or unsubstituted C₁₋₆ alkoxy, C₁₋₆perfluoroalkyl, substituted or unsubstituted straight or branched C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted or unsubstituted C₃₋₁₀cycloalkyl, substituted or unsubstituted C₅₋₈ cycloalkenyl, substitutedor unsubstituted C₁₋₁₀ alkylamino, or substituted or unsubstituted C₅₋₁₀aryl, or substituted or unsubstituted saturated or unsaturated 3-11member heteroaryl or heteroarylalkyl containing 1, 2, 3, or 4heteroatoms selected independently from N, O, S, or S(O)₂;

when Z is N, R₁ is null.

In another aspect, the invention pertains to methods of using compoundsof Formula II in the treatment of various disease states, includingthose involving Bcl-2 family proteins, where Formula II is:

or a stereoisomer thereof, tautomer thereof, solvate thereof, or apharmaceutically acceptable salt thereof, wherein:

X is selected from O, C, or N, with C or N optionally substituted withhydrogen, substituted or unsubstituted C₁₋₆ alkyl, hydroxyl-substitutedalkyl, substituted or unsubstituted C₅₋₁₀ aryl, or substituted carbonylgroup;

R₃ is selected from substituted or unsubstituted C₁₋₆ alkyl, C₁₋₆perfluoroalkyl, substituted or unsubstituted straight or branched C₁₋₁₀alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted or unsubstituted C₃₋₁₀cycloalkyl, substituted or unsubstituted C₅₋₈ cycloalkenyl, substitutedor unsubstituted C₁₋₁₀ alkylamino; and

R₄ is selected from hydrogen, halogen, substituted or unsubstituted C₁₋₆alkyl, C₁₋₆ perfluoroalkyl substituted or unsubstituted straight orbranched C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, substituted orunsubstituted C₃₋₁₀ cycloalkyl, substituted or unsubstituted C₅₋₈cycloalkenyl, substituted or unsubstituted C₁₋₁₀ alkylamino, orsubstituted or unsubstituted C₅₋₁₀ aryl, or substituted or unsubstitutedsaturated or unsaturated 3-11 member heteroaryl or heteroarylalkylcontaining 1, 2, 3, or 4 heteroatoms selected independently from N, O,S, or S(O)₂; and

In another aspect, the invention is directed to the use of compounds ofFormula I or II in treating cancer and autoimmune diseases in patientswith compounds having common substructures or scaffolds identified byanalysis of common structural features of the compounds of Formula I orII, including but not limited to the scaffolds defined in Formula I orII.

In still another aspect, the invention is directed to methods oftreating cancer in patients with compounds of Formula I or II. Cancermay include, for example, hematological malignancies. Such hematologicalmalignancies include, for example, Multiple Myeloma, B-cell lymphoma,acute myelogenous leukemia, and chronic lymphocytic leukemia. Treatmentresults in, for example, tumor regression. Tumor regression can include,for example, killing a cancer cell.

Another aspect of the invention is a method for treating particulartypes of hematopoietic cancers, using a compound of Formula I or II. Theuse of these compounds for particular types of hematopoietic cancershave unexpected results in terms of, for example, efficacy and/orability to inhibit particular anti-apoptotic (pro-survival) members ofthe Bcl-2 family (including, e.g., Mcl-1) or to mimic particular membersof the pro-apoptotic Bcl-2 family proteins. Accordingly, hematologicaltumor cells that are hyper-dependent on a particular member of the Bcl-2family of proteins will be highly impacted by BH3 mimics which targetsthat protein.

In a further aspect, the invention provides a method for killing acancer cell comprising administering an amount of a compound of FormulaI or II effective to kill a cancer cell of a hematopoietic cancer. Thetypes of hematopoietic cancer include, but are not limited to, MultipleMyeloma, B cell lymphoma, chronic lymphocytic leukemia, and acutemyelogenous leukemia.

In another aspect, the invention provides a method for killing a cancercell comprising administering an amount of compound of Formula I or IIin combination with a chemotherapeutic agent or agents that increasesthe level of Mcl-1 in the cancer cell. Such chemotherapeutic agents caninclude 26S proteasome inhibitors and inhibitors of the BH3 domaincontaining E3 ligase called Mule. Such agents may be, but not limitedto, bortezomib or rituximab.

In one embodiment, the invention provides a method for killing a cancercell comprising administering an amount of compound of Formula I or IIin combination with a 26S proteasome inhibitor to kill the cancer cell.A non-limiting exemplary proteasome inhibitor is bortezomib.

In one aspect, the compounds of Formula I or II are used in a method fortreating particular types of hematopoietic cancers, such as B-celllymphoma, to preferentially inhibit the binding of a peptide comprisedof the BH3 domain of Bak to the Bcl-2 family protein Mcl-1. Thisactivity is unique among all of the BH3 mimics reported as of the timeof filing this application and directs the use of this compound intreating certain hematological malignancies that are affectedprincipally by the Bcl-2 family proteins and among those proteins,mostly by Mcl-1. Based on the unique ability of compounds of Formula Ior II to inhibit BH3 binding to Mcl-1, these compounds are useful inblocking the unwanted cell survival activity of Mcl-1 in tumorogeniclymphoid and myeloid cells, and therefore may be used as a therapy fortreating Multiple Myeloma (MM), diffuse large B-cell lymphoma (DLBCL),chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML),all of which are effected by elevated Mcl-1.

In another aspect, the invention provides a method for treatingparticular types of hematopoietic cancers using a combination of one ormore compounds selected from the compounds of Formula I or II, incombination with other therapies, for example, a class of therapeuticsknown as 26S proteasome inhibitors, such as, for example, Bortezomib(VELCADE®).

In another aspect, the invention is directed to methods of treatingautoimmune diseases in patients with compounds of formula identified inFormula I or II. The autoimmune disease may be Type I diabetes,rheumatoid arthritis, osteo arthritis, psoriatic arthritis, psoriasis,neuromyatonia, mayasthenia gravis, lupus erythematosus, endometriosis,Graves disease, granulomatosis, Crohn's disease, interstitial cystitis,or multiple sclerosis, among others.

In another aspect, the invention provides a method for determiningwhether a candidate compound mimics a ligand specific for a target, themethod comprising the steps of (a) providing in a first reaction, thetarget, a first labeled peptide specific for the target, and a firstunlabeled peptide specific for the target, (b) providing in a secondreaction, the target, the first labeled peptide specific for the target,and a first candidate compound, and (c) comparing binding specificity ofthe first unlabeled peptide with binding specificity of the firstcandidate compound to determine whether the candidate compound mimicsthe first unlabeled peptide. In other aspects of the invention, thismethod further comprises repeating steps (a), (b), and (c) wherein thefirst labeled peptide specific for the target is replaced with a secondlabeled peptide specific for the target. In some aspects, the targetcomprises a BH3 domain binding region, such as, for example, ahydrophobic pocket formed by the BH1, BH2, BH3 and BH4 domains of theanti-apoptotic Bcl-2 family of proteins.

In another aspect, the invention provides methods for identifyingspecific activity of BH3 mimic compounds. For example, these compoundscan have varying potencies in inhibiting BH3-mediated binding ofparticular Bcl-2 family proteins, and the difference in potency can beidentified by systematically ordering combinations of protein-proteininteractions and comparing the blocking activity of BH3 mimic compoundsto that of competing BH3 domain containing peptides. By matching theactivity of the compound to a particular BH3 domain peptide, abiological activity can be assigned to that compound that correlates tothe activity of the BH3 domain containing protein. This information canbe used to predict the utility of a BH3 mimic compounds in treating aparticular disease.

In one aspect, the invention provides an agent, which modulatesapoptosis by binding to the Bcl-2 family proteins, including Mcl-1 andpreferentially blocks BH3 domain binding.

In another aspect, the invention provides a method for using a compoundof Formula I or II to preferentially inhibit Mcl-1 over other Bcl-2family members. Particularly, the invention provides a method of usingcompounds of Formula I or II which selectively inhibit Mcl-1 whileshowing dramatically reduced inhibitory activity for Bcl-xL.

In another aspect, the invention provides a method for blocking bindingof the BH3-only Bcl-2 family proteins or parts thereof, including Puma,Noxa, Bim, Bid and Bak, to Mcl-1.

In another aspect, the invention provides a method for using a compoundof Formula I or II as to target Mcl-1 preferentially as inhibitors toinduce apoptosis in cells over expressing Mcl-1.

In another aspect, the invention provides a method for blocking bindingof the BH3-only proteins or parts thereof, including Puma, Noxa, Bim,Bid and Bak, to Mcl-1.

In another aspect, the invention provides a method for using a specificBH3 mimic compound of Formula I or II for inhibiting the activity of theBcl-2 family protein Mcl-1 and other members of the Bcl-2 family ofanti-apoptotic proteins for the purpose of treating cancer and cancerpatients, including those with drug resistance, either alone or incombination with other anti-tumor agents.

In one embodiment, the invention provides a method for using a specificBH3 mimic compound of Formula I or II for inhibiting the activity of theBcl-2 family protein Mcl-1 and other members of the Bcl-2 family ofanti-apoptotic proteins for the purpose of treating lymphoidmalignancies either alone or in combination with other anti-tumoragents.

In another embodiment, the invention provides a method for using aspecific BH3 mimic compound of Formula I or II for inhibiting theactivity of the Bcl-2 family protein Mcl-1 and other members of theBcl-2 family of anti-apoptotic proteins for the purpose of treatingmyeloid cancer either alone or in combination with other anti-tumoragents.

In another embodiment, the invention provides a method for using aspecific BH3 mimic compound of Formula I or II for inhibiting theactivity of the Bcl-2 family protein Mcl-1 and other members of theBcl-2 family of anti-apoptotic proteins for the purpose of treatingprostate cancer either alone or in combination with other anti-tumoragents.

In another embodiment, the invention provides a method for using aspecific BH3 mimic compound of Formula I or II for inhibiting theactivity of the Bcl-2 family protein Mcl-1 and other members of theBcl-2 family of anti-apoptotic proteins for the purpose of treatingnon-Hodgkin's lymphoma patients who are resistant to Rituxan eitheralone or in combination with other anti-tumor agents.

In another embodiment, the invention provides a method for using aspecific BH3 mimic compound of Formula I or II for inhibiting theactivity of the Bcl-2 family protein Mcl-1 and other members of theBcl-2 family of anti-apoptotic proteins for the purpose of treatingChronic Lymphocytic Leukemia patients who are resistant to Rituxaneither alone or in combination with other anti-tumor agents.

In still another embodiment, the invention provides a method for using aspecific BH3 mimic compound of Formula I or II for inhibiting theactivity of the Bcl-2 family protein Mcl-1 and other members of theBcl-2 family of anti-apoptotic proteins for the purpose of treatingbreast cancer either alone or in combination with other anti-tumoragents.

In yet another embodiment, the invention provides a method for using aspecific BH3 mimic compound of Formula I or II for inhibiting theactivity of the Bcl-2 family protein Mcl-1 and other members of theBcl-2 family of anti-apoptotic proteins for the purpose of treatingliver cancer either alone or in combination with other anti-tumoragents.

In another embodiment, the invention provides a method for using aspecific BH3 mimic compound of Formula I or II for inhibiting theactivity of the Bcl-2 family protein Mcl-1 and other members of theBcl-2 family of anti-apoptotic proteins for the purpose of treatingovarian cancer either alone or in combination with other anti-tumoragents.

In another aspect, the invention provides a method for treating cancerpatients with a compound of Formula I or II in combination withproteasome inhibitors.

In one embodiment, the invention provides a method for treatingmyelogenous leukemia with a compound of Formula I or II in combinationwith Bortezomib or other proteasome inhibitors.

In another embodiment, the invention provides a method for treatingchronic lymphocytic leukemia with a compound of Formula I or II incombination with Bortezomib.

In still another embodiment, the invention provides a method fortreating Non-Hodgkin's lymphoma with a compound of Formula I or II incombination with Bortezomib.

In another embodiment, the invention provides a method for treatingbreast cancer with a compound of Formula I or II in combination withBortezomib.

In yet another embodiment, the invention provides a method for treatingprostate cancer with a compound of Formula I or II in combination withBortezomib.

In a further embodiment, the invention provides a method for treatingcolon cancer with a compound of Formula I or II in combination withBortezomib.

In another embodiment, the invention provides a method for treatingpancreatic cancer with a compound of Formula I or II in combination withBortezomib.

In another embodiment, the invention provides a method for treatingliver cancer with a compound of Formula I or II in combination withBortezomib.

In another aspect, the invention provides a method for identifying acompound of the BH3 mimic class of small molecules that is an activeagainst a subset of the BH3 domain containing proteins and therefore haspredicted efficacy against particular tumor types.

In another aspect, the invention provides a method of treating a mammalsuffering from migrating transformed B-cell tumors (non-Hodgkin's)comprising the steps of administering a compound of Formula I or II andmonitoring said mammal to determine the state of said cancer; whereinsaid cancer is a cancer sensitive to said chemical targeted to Bcl-2family proteins; optionally wherein the amount administered is aquantity sufficient to constitute effective treatment, or wherein saidcancer is chosen from a group of cancers comprising: lymphoma, breastcancer, leukemia, lung cancer, bone cancer, prostate cancer gastriccancer, colon cancer, rectal cancer, liver cancer, cervical cancer,renal cancer, bladder cancer, nasopharyngeal cancer, esophagus cancer,pituitary gland tumor, thyroid cancer melanoma, and pancreatic cancer.

In another aspect, the invention provides a method of preventing cancercomprising the step of administering a compound of Formula I or II topersons having a high risk of cancer.

In another aspect, the invention provides a method for selectingspecific activity of a BH3 mimic compound based on similar activity to apeptide comprised of a particular BH3 domain.

In various embodiments of the invention, a mammal is a human. In variousembodiments of the invention, cancer is: Non-Hodgkin's Lymphoma; anyother B-cell lymphomas; Small lymphocytic, consistent CLL; Follicular,predominantly small cleaved cell; Follicular, mixed small cleaved andlarge cell; Intermediate grade Follicular, large cell; Diffuse, smallcleaved cell; Diffuse, mixed small cleaved and large cell; Diffuse,large cell (cleaved and non-cleaved); High grade; Large cell,immunoblastic; Lymphoblastic; Small non-cleaved cell; Burkitt'slymphoma; Non-Burkitt's lymphoma; Indolent NHL; B-cell CLL/smalllymphocytic lymphoma; Marginal zone lymphoma; MALT; Splenic marginal 27.27; zone lymphoma; Nodal marginal zone lymphoma; Lymphomplasmacytoidlymphoma/immunocytoma; Follicle center lymphoma, follicular type Grade I(0-5 centroblasts/hpf) or Grade II (6-15 centroblasts/hpf) or Grade III(>15 centroblasts/hpf); Aggressive NHL; Diffuse, large cell lymphoma;cancer is Mediastinal large cell lymphoma; Primary effusion lymphoma;Mantle cell lymphoma; Burkitt's lymphoma/high-grade Burkitt's-like;Precursor B-cell leukemia/lymphoma; Precursor T-cell leukemia/lymphoma;skin cancer; prostate cancer; gastric cancer; cancer is colon cancer;rectal cancer; liver cancer; cervical cancer; renal cancer; bladdercancer; nasopharyngeal cancer; esophagus cancer; pituitary gland tumor;thyroid cancer; melanoma; small-cell lung cancer; non-small cell lungcancer, or pancreatic cancer.

In various embodiments of the invention, a mammal is a human; cancer isMultiple Myeloma.

In addition, in various embodiments of the invention, a compound of theinvention is administered by injecting it directly into a tumor; acompound of the invention is administered by injecting it into saidmammal's blood stream; a compound of the invention is administeredorally; a compound of the invention is administered through saidmammal's skin; a compound of the invention is administered incombination with chemotherapy agents; or a compound of the invention areadministered in combination with radiation therapy.

In still another aspect, the invention is directed compositions asdefined by Formula I or II. In yet another aspect, the invention isdirected pharmaceutical compositions as defined by Formula I or II.

The details of the invention are set forth in the accompanyingdescription below. Although methods are materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, illustrative methods and materials are now described.Other features, objects, and advantages of the invention will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and features of the invention may be more fully understoodby reference to the drawings as described below in which:

FIG. 1 is a table showing the Mcl-1 and Bcl-xL inhibitory constants(IC₅₀, in μM), obtained as described in Examples 1 and 2, for the smallmolecule Mcl-1 inhibitors compounds 1-8.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates generally to methods and compositions fortreating cancer, including hematological malignancies, such as MultipleMyeloma and B-cell lymphoma, and autoimmune diseases. Further, theinvention relates to treating cancers and autoimmune diseases, with acompound that inhibits the Bcl-2 family protein Mcl-1 as well as otherof the Bcl-2 family proteins. In addition, this invention relates tomethods for determining selectivity of newly classified “BH3 mimic”compounds to predict efficacy in treating hematological and othermalignancies involving Mcl-1.

Definitions

As used herein “anti-apoptotic-protein” is a protein, which whenexpressed in a cell, decreases cell death as compared to a cell thatdoes not express the anti-apoptotic protein. In certain instances celldeath in the cell containing the anti-apoptotic protein is decreased atleast 10% to 90% relative to a control. For instance cell death may bedecreased by about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% and 10%.

As used herein “hematological malignancies” refers to any cancer of theblood or bone marrow, such as leukemia or lymphoma. Examples include,but are not limited to: Myelomas (e.g. Multiple myeloma and Giant cellmyeloma), Acute lymphoblastic leukemia (ALL), Acute myelogenous leukemia(AML), Acute myelogenous leukemia (AML), Chronic myelogenous leukemia(CML), Acute monocytic leukemia (AMOL), Hodgkin's lymphomas (e.g., allfour subtypes). In addition, Non-Hodgkin's lymphomas such as Diffuselarge B-cell lymphoma (DLBCL), Follicular lymphoma (FL), Mantle celllymphoma (MCL), Marginal zone lymphoma (MZL), Burkitt's lymphoma (BL),Burkitt's lymphoma (BL), and other NK- or T-cell lymphomas are included.

As used herein, the term “Bcl-2” refers to the protein originallydiscovered as the causal “oncogene” in lymphomas.

As used herein “pro-apoptotic protein” means a protein that whenexpressed in a cell increases cell death, as compared to a cell thatdoes not express the pro-apoptotic protein. In certain instances celldeath in the cell containing the pro-apoptotic protein is increased atleast 10% to 90% relative to a control. For instance cell death may beincreased by about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% and 10%.

By “disrupts an interaction” it is meant that a test compound decreasesthe ability of two polypeptides to interact with each other. In certaininstances the disruption results in at least a 99% decrease in theability of the polypeptides to interact with each other. Disruptions canbe identified using a combination of virtual screening for molecularstructures, which fit the ideal structure of BH3 pocket, and competitionbinding studies using fluorescence polarization (FP).

“Fluorescence polarization assay” means an assay in which an interactionbetween two polypeptides is measured. In this assay, one polypeptide islabeled with a fluorescent tag, and this polypeptide emits non-polarizedlight when excited with polarized light. Upon an interaction of thetagged polypeptide with another polypeptide, the polarization of emittedlight is increased, and this increased polarization of light can bedetected.

“Interacts” means a compound that recognizes and binds to ananti-apoptotic protein but which does not substantially recognize andbind to other molecules in a sample.

“Alkyl” refers to a hydrocarbon chain that may be a straight chain orbranched chain, containing the indicated number of carbon atoms. Forexample, C₁-C₁₀ indicates that the group may have from 1 to 10(inclusive) carbon atoms in it.

“Hydroxyl-substituted alkyl” refers to a hydrocarbon chain that may be astraight chain or branched chain, containing the indicated number ofcarbon atoms, in which at least one of the hydrogen atoms has beensubstituted with an OH group.

“Aryl” refers to an aromatic hydrocarbon group. If not otherwisespecified, in this specification the term aryl refers to a C₆-C₁₄arylgroup. Examples of an C₆-C₁₄aryl group include, but are not limited to,phenyl, 1-naphthyl, 2-naphthyl, 3-biphen-1-yl, anthryl,tetrahydronaphthyl, fluorenyl, indanyl, biphenylenyl, and acenaphthenyl,groups. An aryl group can be unsubstituted or substituted with one ormore of the following groups: C₁-C₆alkyl, C₃-C₁₀cycloalkyl,C₁-C₆perfluoroalkyl-, halo, haloalkyl-, hydroxyl, C₁-C₆hydroxyalkyl-,—NH₂, aminoalkyl-, dialkylamino-, —COOH, carboxylic ester, primary,secondary, or tertiary carboxylic amide, urea, —(C(O)—(C₁-C₆alkyl),—OC(O)(C₁-C₆alkyl), N-alkylamido-, —C(O)NH₂, (C₁-C₆alkyl)amido-,—S(O)₂N-alkyl, —S(O)₂N-aryl, alkoxy, or —NO₂.

“Alkoxycarbonyl” refers to the group C(O)—OR where R is an alkyl group,as defined above.

“Aryloxycarbonyl” refers to the group C(O)—OR where R is an aryl group,as defined above.

“Alkoxy” refers to the group R—O— where R is an alkyl group, as definedabove. Exemplary C₁-C₆alkoxy groups include but are not limited tomethoxy, ethoxy, n-propoxy, 1-propoxy, n-butoxy and t-butoxy.

“Perfluoroalkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms, in which all hydrogen atoms have been substituted with fluorines.

“Alkenyl” refers to a straight or branched chain unsaturated hydrocarboncontaining at least one double bond.

“Alkynyl” refers to a straight or branched chain unsaturated hydrocarboncontaining at least one double bond.

“Cycloalkyl” refers to a hydrocarbon ring containing the indicatednumber of carbon atoms.

“Cycloalkenyl” refers to a hydrocarbon ring containing the indicatednumber of carbon atoms and at least one double bond.

“Aminoalkyl” refers to an alkyl group, as defined above, wherein one ormore of the alkyl group's hydrogen atoms has been replaced with N. ThisN may be further substituted with alkyl groups, aryl groups, heteroarylgroups, heteroarylalkyl groups, or aminoalkyl groups.

“Alkylamino” refers to a N which is connected to an alkyl group, asdefined above. This alkyl group may be branched or unbranched or furthersubstituted with aryl groups, heteroaryl groups, heteroarylalkyl groups,or aminoalkyl groups.

“Hydroxyl” refers to an OH group.

“Heteroaryl” refers to mono, bicyclic, and tricyclic aromatic groups of5 to 10 atoms containing at least one heteroatom and at least onearomatic ring. Heteroatom as used in the term heteroaryl refers tooxygen, sulfur and nitrogen. Examples of monocyclic C₃-C₉ heteroarylsinclude, but are not limited to, pyrrolyl, oxazinyl, thiazinyl,pyridinyl, diazinyl, triazinyl, tetrazinyl, imidazolyl, tetrazolyl,isoxazolyl, furanyl, furazanyl, oxazolyl, thiazolyl, thiophenyl,pyrazolyl, triazolyl, and pyrimidinyl. Examples of bicyclic C₄-C₁₀heteroaryls include but are not limited to, benzimidazolyl, indolyl,indolinyl, isoquinolinyl, quinolinyl, quinazolinyl, benzothiophenyl,benzodioxolyl, benzo[1,2,5]oxadiazolyl, purinyl, benzisoxazolyl,benzoxazolyl, benzthiazolyl, benzodiazolyl, benzotriazolyl, isoindolyland indazolyl.

“Aminocarbonyl” refers to the group NC(O)R, wherein R may be alkyl,aryl, or heteroaryl, as defined above.

“Aminosulfonyl” refers to the group NS(O)₂R, wherein R may be alkyl,aryl, or heteroaryl, as defined above.

“Halo” refers to the groups F, Cl, or Br.

“Heteroarylalkyl” refers to an alkyl group, as defined above, whereinone or more of the alkyl group's hydrogen atoms has been replaced with aheteroaryl group as defined above.

The “unsubstituted or substituted” refers to a non-replacement orreplacement of one or more hydrogen atom in the indicated radical withone or more of the following groups: C₁-C₆alkyl, C₃-C₁₀cycloalkyl,C₁-C₆perfluoroalkyl-, halo, haloalkyl-, hydroxyl, C₁-C₆hydroxyalkyl-,—NH₂, aminoalkyl-, dialkylamino-, —COOH, carboxylic ester, primary,secondary, or tertiary carboxylic amide, urea, —C(O)O—(C₁-C₆alkyl),—OC(O)(C₁-C₆alkyl), N-alkylamido-, —C(O)NH₂, (C₁-C₆alkyl)amido-,—S(O)₂N-alkyl, —S(O)₂N-aryl, alkoxy, or —NO₂. For example “substitutedalkyl” refers to an alkyl group where one or more hydrogen atom isindependently replaced by one or more of the above groups.

“Substituted carbonyl” refers to a carbonyl group (—C═O) furthersubstituted with OH, NH₂, SH, halogen, or C₁-C₆alkyl.

The present invention relates to compositions and methods for thetreatment of cancer and autoimmune disease. More specifically, thepresent invention relates to compositions and methods for treatinghematological malignancies. Such hematological malignancies include, forexample, Multiple Myeloma, lymphoma, acute myelogenous leukemia, andchronic lymphocytic leukemia. Such treatment, results in, for example,tumor regression in a mammal, such as a mouse or a human. Tumorregression can include, for example, killing a cancer cell.

The invention also relates to compounds of Formula I or II.

The invention also relates to treating hematological malignancies with acompound of Formula I or II, and/or a BH3 mimic compound that inhibits abroad range of the Bcl-2 family of proteins, most notably Mcl-1. It iscontemplated that the activity against the protein Mcl-1 of a compoundof Formula I or II as well as derivative compounds will enabletherapeutic utility of these compounds as anti-tumor agents in treatingcancer, including blood cancers.

The invention, for example, provides a method for treating particulartypes of hematopoietic cancers, using a BH3 mimic compound of Formula Ior II. The use of these compounds for particular types of hematopoieticcancers may have unexpected results in terms of efficacy and/or abilityto inhibit particular anti-apoptotic (pro-survival) members of the Bcl-2family or to mimic particular members of the pro-apoptotic Bcl-2 familyproteins. Accordingly, hematological tumor cells that arehyper-dependent on a particular member of the Bcl-2 family of proteinsmay be highly affected by that BH3 mimic which targets that protein.

A compound Formula I or II may be particularly useful in a method oftreating hematopoietic cancers, by preferentially inhibiting the bindingof the activator BH3 only proteins of the Bcl-2 family to protein Mcl-1.The high affinity of these compounds towards MCL-1 (<20 μM) directs theuse of these compounds in treating certain hematological malignanciesthat are affected principally by the Bcl-2 family proteins and amongthose proteins, mostly by Mcl-1. Based on the unique ability of acompound of Formula I or II to inhibit BH3 binding to Mcl-1, thesecompounds may be particularly effective in blocking tine unwanted cellsurvival activity of Mcl-1 in tumorogenic lymphoid and myeloid cells.This feature of compounds of Formula I or II direct their use as apotential therapeutic agents for treating Multiple Myeloma (MM), diffuselarge B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), acutemyelogenous leukemia (AML), all of which are effected by elevated Mcl-1.Similar activity in derivatives of compounds of Formula I or II, maydirect the use of those compounds in treating lymphoid and myeloidmalignancies.

This invention also relates to using a compound of Formula I or II tocause tumor regression and enhance survival in B-cell lymphomas.Accordingly, in one embodiment, this invention describes a method forusing a compound of Formula I or II for the treatment of non-Hodgkin'sB-cell lymphoma, including CLL, Burkett's Indolent and Aggressivenon-Hodgkin's lymphomas, Multiple Myelomas, or other cancers that areaffected by Bcl-2 family of proteins, and in particular the proteinMcl-1.

These treatments may be accomplished utilizing a compound of Formula Ior II alone, or in combination, with other chemotherapy agents or withradiation therapy. Accordingly, the invention provides a method fortreating particular types of hematopoietic, cancers using a combinationof one or more compounds of Formula I or II, in combination with othertherapies, for example, a class of therapeutics known as 26S proteasomeinhibitors. In some embodiments, the 26S proteasome inhibitor isBortezomib (VELCADE®).

In addition, this invention relates to methods for determiningselectivity of a compound of Formula I or II and BH3 mimic compounds topredict efficacy in treating hematological and other malignanciesinvolving Bcl-2 family proteins. For example, these compounds can havevarying potencies in inhibiting BH3 mediated-binding of particular Bcl-2family proteins, and the difference in potency can be identified bysystematically ordering combinations of protein-protein interactions andcomparing the blocking activity of BH3 mimic compounds to that ofcompeting BH3 domain-containing peptides. By matching the activity ofthe compound to a particular BH3 domain peptide, a biological activitycan be assigned to that compound that correlates to the activity of theBH3 domain-containing protein. This information can be used to predictthe utility of a BH3 mimic compounds in treating a particular disease.

This invention also relates to cancer treatments, Multiple Myelomatreatments directed to Bcl-2 and Bcl-xL and Mcl-1 and A1 and Bcl-w(referred as a group as an anti-apoptotic Bcl-2 family) activity.

The present invention also relates to determining a cellular state withrespect to programmed cell death. This state has been named “primed fordeath”. In addition to alive and dead states, cells may be primed fordeath in that they require tonic antiapoptotic function for survival.

Previous work demonstrated a method called BH3 profiling (Certo, et al.(2006) Cancer Cell 9(5): 351-65; Deng, et al. (2007) Cancer Cell. 12(2):171-85; U.S. Patent Publication No. 2008/0199890). This method uses apanel of peptides derived from BH3 domains of BH3-only proteins thatselectively antagonize individual BCL-2 family members BCL-2, BCL-XL,BCL-w, MCL-1 and BFL-1. It was shown that cellular “addiction” toindividual antiapoptotic proteins can be diagnosed based onmitochondrial response to these peptides. This panel of peptides isshown in Table 1 and are referred to herein as BH3 domain peptides.Antiapoptotic proteins BCL-2, BCL-XL, MCL-1, BFL-1 and BCL-w each bear aunique pattern of interaction with this panel of proteins. Cellulardependence on an antiapoptotic protein for survival is decoded based onthe pattern of mitochondrial sensitivity to this peptide panel. Thisstrategy is called BH3 profiling.

TABLE 1 BH3 Domain Peptides Amino Acid Sequence SEQ ID NO BIDEDIIRNIARHLAQVGDSMDR  1 BIM MRPEIWIAQELRRIGDEFNA  2 BID mutEDIIRNIARHAAQVGASMDR  3 BAD LWAAQRYGRELRRMSDEFEGSFKGL  4 BIKMEGSDALALRLACIGDEMDV  5 NOXA A AELPPEFAAQLRKIGDKVYC  6 NOXA BPADLKDECAQLRRIGDKVYC  7 HRK SSAAQLTAARLKALGDELHQ  8 BNIPVVEGEKEVEALKKSADWVSD  9 PUMA EQWAREIGAQLRRMADDLNA 10 BMFHQAEVQIARKLQLIADQFHR 11 huBAD NLWAAQRYGRELRRMSDEFVDSFKK 12 BAD mutLWAAQRYGREARRMSDEFEGSFKGL 13

Mitochondria are probed to determine a cell's state using this panel ofsensitizer BH3-peptides, selective antagonists of antiapoptotic BCL-2family members. Mitochondria that are primed for death are dependent onantiapoptotic protein function to prevent mitochondrial outer membranepermeabilization (MOMP), so that they release cytochrome c when exposedto sensitizer BH3 peptides. In contrast, unprimed cells do not releasecytochrome c when exposed to sensitizer BH3 peptides. Any cell fromwhich mitochondria can be isolated can therefore be so tested andcategorized as being primed or imprinted. A “snapshot” of the apoptoticstate at a given time may be taken with minimal perturbation of theextant apoptotic machinery. In summary, BH3 profiling allows capture ofinformation about a fundamental aspect of cellular physiology.Importantly, mitochondrial behavior is correlated to whole cell behaviorin several models, and BH3 profiling revealed a dependence onantiapoptotic proteins only when a cellular dependence was alsodemonstrated.

Not all cells are sensitive to antagonism of antiapoptotic proteins.Sensitive cells are “primed for death” with death signals carried by aselect subset of proapoptotic proteins of the BCL-2 family. Some cancercells may be tonically primed for death, and thus are selectivelysusceptible to agents that provoke or mimic sensitizer BH3-only domains.It has been postulated that inhibition of apoptosis is a requirement ofoncogenesis (Green and Evan, (2002) Cancer Cell. 1(1):19-30; Hannahanand Weinberg, (2002) Cell. 100(1)57-70). In what may be an attempt tomeet this requirement, many types of cancer cells overexpressantiapoptotic BCL-2 family members. Understanding how these proteinsfunction is therefore critical to understanding how cancer cellsmaintain survival.

The present invention provides a method for determining the “primed todie” state of a cell without using peptides. This method allows theinvestigation of whether a particular antiapoptotic BCL-2 family member,Mcl-1, controls mitochondrial outer membrane permeabilization (MOMP) andcommitment to apoptosis. Antiapoptotic proteins show selective affinityfor binding BH3 peptides derived from BH3-only proteins. For example,Mcl-1 binds selectively to the BH3 peptide Noxa. Furthermore, antagonismof Mcl-1 by Noxa results in MOMP only when Mcl-1 is “printed” withactivator BH3 proteins, validating the critical role of activator BH3domains in activating BAX/BAK. In cell culture models, activator“priming” can be observed following experimentally-induced deathsignaling, and that such priming confers dependence on antiapoptoticfamily members. The dependence on antiapoptotic BCL-2 family members canbe captured functionally by the pattern of mitochondrial sensitivity tosensitizer BH3 domains, and Mcl-1 dependence can be identified bysensitivity to Noxa. Accordingly, the invention includes methods ofdetermining the sensitivity of a cell to a class of therapeutic agents,Mcl-1 inhibitors. This method identifies whether or not a cell is primedfor death through an Mcl-1 dependent mechanism by measuringmitochondrial sensitivity to the Mcl-1 specific inhibitor compounds ofFormula I or II.

Compounds that exhibit a high degree (>10-fold) of selectivity andspecificity for any specific Bcl-2 family protein can be used identifywhether a cell is primed for death via a mechanism that involves otherBcl-2 family proteins. The compounds ABT-737 and ABT-263 (Tse, et al.(2008) Cancer Res. 68, 3421-3428) are highly specific to Bcl-2 andBcl-xL (IC₅₀<10 nM versus Bcl-2 and Bcl-xL) but much weaker binders toMcl-1 (IC₅₀<500 nM) and can be used to treat cancer cells ormitochondria from cancer cells in order to determine if the cell isprimed with Bcl-2 and/or Bcl-xL and to determine if the cancer cell issensitive to Bcl-2 and/or Bcl-xL inhibition. In a similar way, compoundsof this invention that are specific to the Bcl-2 family protein Mcl-1may be used to treat cancer cells or mitochondria from cancer cells inorder to determine if the cancer cell is sensitive to Mcl-1 inhibition.

As a chemical probe BH3 mimetic this compound can be used for diagnosticpurposes. Response of isolated or in situ cancer cell mitochondria tothis compound could indicate a patient being predisposed to treatmentwith appropriately targeted therapeutic compounds.

Additional aspects of this approach are discussed in U.S. PatentPublication Nos. 2008-0199890 and 2009-0280510 and U.S. Pat. No.7,868,133, the contents of which are incorporated herein by reference intheir entirety.

There is provided in accordance with one aspect of the invention,compounds of Formula I. Also provided are pharmaceutical compositions ofFormula I and methods of using compounds of Formula I. Formula I is:

-   -   and stereoisomers thereof, tautomers thereof, solvates thereof,        and pharmaceutically acceptable salts thereof, wherein:

X, Y, Z, Z′, R₁, and R₂ are defined as above for Formula I.

In some embodiments, X is NEt.

In other embodiments, X is NCO₂tBu.

In other embodiments, X is O.

In some embodiments, Y is CH.

In some embodiments, Z is C.

In some embodiments, Z′ is CH.

In some embodiments, R₁ is trifluoromethyl.

In other embodiments, R₁ is fluoro.

In other embodiments, R₁ is methoxy.

In other illustrative embodiments, compounds of Formula I are set forthbelow:

-   6-((4-ethylpiperazin-1-yl)(4-(trifluoromethyl)phenyl)methyl)isoquinolin-5-ol    (1);-   tert-butyl    4-((4-fluorophenyl)(5-hydroxyisoquinolin-6-yl)methyl)piperazine-1-carboxylate    (2);-   6-((4-ethylpiperazin-1-yl)(4-methoxyphenyl)methyl)isoquinolin-5-ol    (3);-   6-((4-ethylpiperazin-1-yl)(4-fluorophenyl)methyl)isoquinolin-5-ol    (4);-   6-(morpholino(4-(trifluoromethyl)phenyl)methyl)isoquinolin-5-ol (5);-   6-((4-fluorophenyl)(morpholino)methyl)isoquinolin-5-ol (6), and;-   6-((4-methoxyphenyl)(morpholino)methyl)isoquinolin-5-ol (7).

In another aspect, there is provided compounds of Formula II. Alsoprovided are pharmaceutical compositions of Formula II and methods ofusing compounds of Formula II. Formula II is:

-   -   and stereoisomers thereof, tautomers thereof, solvates thereof,        and pharmaceutically acceptable salts thereof, wherein:

X, R₃, and R₄, are defined as above for Formula II.

In some embodiments, X is O.

In some embodiments, X is NEt.

In some embodiments, R₃ is methyl.

In some embodiments, R₄ is trifluoromethyl.

In other illustrative embodiments, compounds of Formula II are set forthbelow:

-   7-((4-ethylpiperazin-1-yl)(4-(trifluoromethyl)phenyl)methyl)-8-triethoxyquinoline    (8);-   7-((4-ethylpiperazin-1-yl)(4-fluorophenyl)methyl-8-methoxyquinoline    (9), and;-   4-((4-fluorophenyl)(8-methoxyquinolin-7-yl)methyl)morpholine (10).

Methods of Using A. Treatment with a Compound of Formula I or II toInhibit Bcl-2 Proteins

The present invention describes, in part, anti-tumor efficacy andenhanced survivability in mouse models for hematological malignancies bytreatment with a compound of Formula I or II. Efficacy in certain animalmodels may be correlated to humans afflicted with B-cell lymphoma orother hematological or non-hematological cancers affected by Bcl-2family proteins.

This treatment may be administered as a stand-alone therapy, or withother chemotherapy agents, or with radiation therapy. In one embodiment,a compound of Formula I or II is used for the treatment of B-celllymphoma or Multiple myeloma by inducing cancer cell death andpreventing cancer cell migration to spleen or lymph nodes.

Because Mcl-1 has emerged as a key member of the Bcl-2 family ofproteins for initiating and maintaining certain myeloid as well asB-cell and T-cell malignancies, it is an important target for treatmentof many hematological diseases. This invention demonstrates, amongothers, the effectiveness of a compound of Formula I or II in inhibitingBH3 binding to Mcl-1. Thus, these compounds are useful for treatingMultiple Myeloma, B-cell lymphoma or other hematological cancers orother disease that are affected by Mcl-1 activity including prostate,liver, and ovarian cancers.

In some embodiments, the compounds of Formula I or II have activityagainst Mcl-1, therefore the compounds of Formula I or II and/or otherBH3 mimic compounds against Mcl-1 are useful therapeutic compounds intreating MM, NHL, CLL, AML and prostate, liver, and ovarian cancers,among others.

This activity will also direct the use of these compounds for treatmentof certain autoimmune diseases that are affected by excess B or T cellproliferation.

The present invention relates to the use of a compound of Formula I orII, as compositions in inhibiting the activity of Bcl-2 pro-survivalproteins, most particularly Mcl-1, in tumor cells and thereby killingthose cells. The unique ability of these compounds to inhibit Mcl-1function in cells will makes these compounds effective anti-B-cell,T-cell, and myeloma cell cancer therapeutics for treating non-Hodgkin'slymphoma, CLL, MM, and AML as well as prostate, colon, ovarian, andliver cancer, among other diseases.

A compound of Formula I or II, causes tumor regression, for example bykilling a cancer cell, and increased survival in several mouse tumormodels, including, for example, models for diffuse large B-cell lymphoma(DLBCL) (Cattoretti, et al. (2005) Cancer Cell 7: 445-55), small B celllymphoma/CLL (Zapata, et al. (2004) Proc. Natl. Acad. Sci. USA 101(47):16600-5) and migrating B-cell lymphomas (Refaeli, et al. (3018) Proc.Natl. Acad. Sci. USA 102(11): 4097-102), as well as an AML mouse tumormodel (Lopes de Menezes, et al. (2005) Clin. Canc. Res. 11(14):5281-91). All of the tumors from these cell models are characterized ashaving elevated pro-survival Bcl-2 family proteins, including Bcl-2,Bcl-xL, and Mcl-1.

Accordingly, the present invention relates to the use of a compound ofFormula I or II, in affecting tumor regression in human lymphoid andmyeloid cancers. These compounds are effective in inducing apoptosisselectively in hematological cancers due to the hyper-dependence oflymphoid and myeloid-derived tumor cells on the activity of the Bcl-2family anti-apoptotic proteins.

The Bcl-2 protein is a member of an entire family, the Bcl-2 family ofproteins, that have structurally similar genes and that share sequencehomology and participate in the control of programmed cell death or“apoptosis” (Corey, et al. (2002) Nat. Rev. Cancer 2: 647-656). Somemembers of this family (anti-apoptotic Bcl-2 family proteins), such asBcl-2 Bcl-xL, BcL-w, Bfl-1(A1) and Mcl-1, protect cells from apoptosis.These proteins share sequence homology in four α-helical regions calledthe Bcl-2 homology (BH)-domains 1-4 (BH1-BH4). Another class of thisfamily (pro-apoptotic Bcl-2 proteins), such as Bax and Bak, promoteapoptosis and share three of these domains, BH1-BH3. A third class ofBcl-2 family proteins, such as Bim, Bad, Hrk, Bid, Puma, Noxa, and Bmf,share only one region, the BH3 domain, and are referred to as “BH3-onlyproteins.” The BH3-only proteins are pro-apoptotic, and like Bax andBak, the BH3-only proteins require an intact BH3 domain to promoteapoptosis (Adams, et al. (1998) Science 281: 1322-26).

A complex interplay of the pro-apoptotic and anti-apoptotic proteinsaffects the integrity of the outer membrane of the mitochondria (Green,et al. (2004) Science 305: 626-29) either causing or preventing therelease of certain molecules that activate the cystein aspartylproteases (caspases). The caspases are the eventual effectors ofapoptosis (Salvesen (2002) Cell Death and Differentiation 9: 3-5). Baxand Bak are essential for release of these apoptosis promoting moleculesfrom the mitochondria (Wei, et al. (2001) Science 292: 727-30). TheBH3-only proteins stimulate the activity of Bax and Bak while theanti-apoptotic proteins oppose their activity. Essentially all of theseinteractions occur by BH3 domain mediated binding (Chrittenden, et al.(1995) EMBO J. 14: 5589-96).

Anti-apoptotic family members Bcl-2, Bcl-xL and Mcl-1 are over-expressedin many types of cancers, including lymphomas, melanomas, myelomas, andcancer in the prostate and colon (Kitada, et al. (2002) Oncogene 21:3459-74; Paul-Samojedny, et al. (2005) Biochem. Biophys. Acta.1741(1-2): 25-29; Pollack, et al. (2003) Cancer 97(7): 1630-8; Tas, etal. (2004) Melanoma Res. 14(6): 543-6). Animal model studies establishedthat the continuous presence of anti-apoptotic family members isrequired for tumor survival and growth. Additionally, the pro-survivalBcl-2 proteins are important for the development of resistance of tumorcells to chemotherapies such as DNA damaging agents. The ratio ofpro-apoptotic to anti-apoptotic family members has been shown in manycases to hold significant prognostic value for patient outcome.Over-expression of anti-apoptotic Bcl-2 family proteins has beenreported in many of the hematopoietic malignancies. For example,increased expression of Bcl-2 protein that results from a translocation(t14; 18) of the BCL2 gene occurs in 80% to 90% of low-grade follicularnon-Hodgkin lymphomas (NHLs) (Kitada, et al. (2002) supra).

Three different strategies for countering the tumorigenic effects ofanti-apoptotic Bcl-2 family proteins in NHL, CLL, MM, and other types ofcancer include: (1) inhibiting gene transcription; (2) using antisenseoligonucleotides to cause mRNA degradation; and (3) directly inhibitingthe proteins with small-molecule drugs (reviewed in Reed, et al. (2005)Blood 106: 408-418).

One of the desired characteristics of anti-tumor drugs is the ability toinduce apoptosis in tumor cells and not in healthy cells. Conventionalchemotherapy is mostly based on the evidence that proliferating cellsare more sensitive to anticancer agents than non-dividing cells(Marchini, et al. (2004) Curr. Med. Chem. Anticancer Agents 4(3):247-6). For instance, tumor cells are generally more sensitive toapoptosis induction by microtubule poisons such as taxol andDNA-damaging drugs such as doxorubicin, than healthy cells (Abal, et al.(2003) Curr. Cancer Drug Targets 3(3): 193-203).

However, in many types of cancer, levels of certain members of theanti-apoptotic Bcl-2 family proteins are elevated which causes cells tobe less responsive to such drugs. This is especially true in B-celllymphomas and other hematological malignancies. In these cancerselevated levels of anti-apoptotic Bcl-2 family proteins correlate highlywith the onset of disease, maintenance of the disease state, andchemoresistance (Kitada et al. (2002) supra).

However, it was reported that cells over-expressing Bcl-xL exhibitedincreased sensitivity to an antimycin-A derivative compound that bindsto and inhibits Bcl-2 and Bcl-xL (Manion, et al. (2004) J. Biol. Chem.279(3): 2159-65; Kim, et al. (2001) Biochemistry 40: 4911-22). Thisfinding has implications for the use of certain BH3 mimics as anti-tumortherapeutic compounds given that over-expression of Bcl-xL or Bcl-2results in a general decrease in responsiveness to apoptotic cues andhas been implicated in multi-drug resistance in cancer cells andcarcinogenesis.

An understanding of the mechanisms for this observed change in responseto Bcl-2 or Bcl-xL targeted compounds has been described (Letai, (2005)J. Clin. Invest. 115: 2648-55). In that report it was argued that thecell context in which elevated anti-apoptotic Bcl-2 proteins are founddetermines the occurrence or the degree of “sensitization” to apoptoticcues. Most notably, it is the presence of BH3-only proteins bound tothese anti-apoptotic proteins that cause sensitization to apoptoticcues. For instance, the presence of the BH3-only protein Bad (Bcl-2associated death promoter) bound to Bcl-2 or Bcl-xL sensitizes ratherthan kills cells, as is the case when the BH3-only protein Bim (Bcl-2like 11) binds (Letai, et al. (2002) Cancer Cell 2(3): 183-92). Otherarguments have been put forth that describe “hyper-dependence” oncertain elevated anti-apoptotic Bcl-2 proteins in certain tumor cells(Kim, et al. (2001) Biochemistry 40: 4911-22).

The functions that the individual Bcl-2 family proteins have duringhematopoiesis have been demarcated genetically using transgenic mice.For example, mice deficient in Bcl-2 have no overt problems duringlymphocyte differentiation but do have excess apoptosis in peripherallymphocytes after antigenic stimuli (Veis, et al. (1993) Cell 75: 229).Bcl-xL deficient mice are also viable but do show late maturation oferythroid cells (Wagner, et al. (2000) Development 127: 4949-58).

Mcl-1 deficiency has a pronounced, perhaps principal role in lymphocytesurvival. Conditional knockouts have been used to determine the role ofMcl-1 in hematopoiesis and lymphocyte survival. Conditional deficiencyof Mcl-1 results in apoptosis of differentiating lymphocytes and stopsdevelopment of pre-B-cell and double negative T-cells as well asapoptosis in mature B and T lymphocytes (Rinkenberger, et al. (2000)Genes Dev. 14: 23). Thus the anti-apoptotic form of Mcl-1 plays a rolein the development and survival of B and T lymphocytes, and may be anideal target for treating excess growth of lymphoid cells.

The clinical implication is underscored by the observation that elevatedMcl-1 expressed in its active anti-apoptotic full length form positivelycorrelates with increasing grade of B-cell lymphomas and plasma cellmyelomas (Cho-Vega, et al. (2004) Hum. Pathol. 35 (9): 1095-100) as wellas chronic lymphocytic leukemia (Petlickovski, et al. (2005) Blood 105:4820-28).

Targeted gene knockouts for different pro-apoptotic BH3-only members ofthe Bcl-2 family members have been assessed for disease correlation.Transgenic mice deficient in Bim have extensive myeloid proliferationand autoreactive T and B cells that have lost responsiveness toapoptosis inducing drugs (Bouillet, et al. (1999) Science 286: 1735-38),while mice deficient in Bad display high incidence of diffuse large celllymphoma (Ranger, et al. (2003) Proc. Natl. Acad. Sci. USA 100:9324-29). Mice deficient in the pro-apoptotic BH3-only protein Biddemonstrated Hepatocarcinoma and a failure to respond to the deathinducing cytokine fas (Yin, et al. (1999) Nature 400: 886-891). Both ofthe pro-apoptotic BH3-only proteins Puma and Noxa were shown to berequired for all p53-mediated apoptosis (Villunger, et al. (2003)Science 302: 1036-1040).

Notably, conditional knockouts of the Mcl-1 gene caused profoundreduction in B and T lymphocytes (Opferman, et al. (2003) Nature426(6967): 671-6), which is the opposite of a deficiency in the BH3-onlyprotein Bim and in keeping with the understanding that Mcl-1 selectivelyinhibits the pro-apoptotic protein Bim.

B. Combination Therapy

Embodiments of the present invention also include the combination of oneor more compounds of Formula I or II with other anti-tumor agents, suchas proteasome inhibitors, to yield combination therapies. In someinstances, these combination therapies may yield synergistic results ascompared to the additive results of the component therapies when usedalone. For example, these compounds may be particularly effective whenused in combination with a class of therapeutics known as 26S proteasomeinhibitors.

Compounds that have activity as 26S proteasome inhibitors have beensuggested for use as anti-tumor therapeutics based on their ability toinhibit NF-κB signaling (Li, et al. (1995) Biophys. Biochem. Res. Com.215: 292-301). One such compound, the FDA approved drug Bortezomib(VELCADE®), has been shown to cause elevated Mcl-1 in lymphocytes(Nencioni, et al. (2005) Blood 105(8): 3255-62). Elevated Mcl-1 has beenshown to be causal in the establishment and maintenance of lymphoid andmyeloid tumors. Unwanted side effect of elevated. Mcl-1 can be rectifiedby inhibiting Mcl-1 using the compounds of Formula I or II, and thatthese compounds will have utility in potentiating the effect ofBortezomib or other 26S proteasome inhibitors as anti-tumortherapeutics.

Proteasome inhibitors exhibit anti-tumor activity against malignanciesof different histology. The rationale for looking at proteasomeinhibitors as cancer therapeutics comes from the understanding thatNF-kB is blocked by IkB which, following phosphorylation andubiquination, is degraded in the 26S proteasome (Li, et al. (1995)supra). Following the degradation of IkB, NF-kB translocates to thenucleus where it functions as a transcription factor. NF-kB activatestranscription of growth-promoting genes such as the interleukins as wellas anti-apoptosis protein IAP and Bcl-2 (Wang, (1998) Science 281:1680-83; Fahy, (2005) Cancer Chemother. Pharmacol. 56(1): 46-54).Blocking the 26S proteasome and degradation of then becomes an approachfor inhibiting the growth-promoting and anti-apoptotic effects of NF-κB.

Empirical findings have indicated that actively proliferating cells aremore sensitive to proteasome blockade than quiescent cells. For examplehuman chronic lymphocytic leukemia (CLL) cells are much more sensitiveto the proteasome inhibitor lactacystin than are normal lymphocytes.(Masdehors, et al. (1999) Br. J. Haematol. 105: 752-57). Oral squamouscarcinoma cells are also more sensitive to lactacystin than normal oralepithelial cells (Kudo, et al. (2000) Clin. Cancer Res. 6: 916-923). Theproteasome inhibitor Bortezomib or PS-341 or VELCADE® (Hideshima, et al.(2001) Cancer Res. 61: 3071-76) has a more pronounced effect on humanmultiple myeloma compared to peripheral-blood mononuclear cells. Anotherproteasome inhibitor MG-132 demonstrates preferential killing of acutemylogenous leukemia (AML) cells over normal CD34+ cells (Guzman, et al.(2002) Proc. Natl. Acad. Sci. 99: 16220-25). Currently, the MG-132compound is in pre-clinical studies while MLN-519, a lactacystinsynthetic derivative, in phase 1 clinical trials. The therapeuticBortezomib (VELCADE®; Millennium Pharmaceuticals, and Johnson & JohnsonPharmaceutical Research & Development) has been approved for use intreatment of multiple myeloma. Introduced in the U.S. in 2003, VELCADEprovides an improvement in MM therapy, and is approved as a first linetherapy in combination with oral melphalan and prednisone. VELCADE isnow widely prescribed in combination with dexamethasone (Doxil,Adriamycin) or with dexamethasone and the thalidamide drugs Revlicade(lenolidomide) and Thalidomid (thalidomide), the so-called RVDtreatment.

Recent evidence indicates that some anti-apoptotic factors accumulate asa consequence of exposure to Bortezomib (VELCADE®), possibly reducingits effectiveness as an anti cancer therapeutic. Most notably the Bcl-2family member Mcl-1 is elevated in cells treated with Bortezomib(VELCADE®) (Nencioni, et al. (2005) Blood 105(8): 3255-62). This isproblematic as the proteasome-mediated reduction of Mcl-1 is aninitiating signal for apoptosis in response to genotoxic stimuli(Cuconati, et al. (2003) Genes Dev. 17(23): 2922-32) and antigenreceptor signaling in B-cells (Petlickovsky, et al. (2005) Blood105(12): 4820-7). Further, sustained signaling through the B-cellreceptor induces Mcl-1 and promotes survival of chronic lymphocyticleukemia B cells (Petlickovsky, et al. (2005) supra).

These observations suggest that elevated Mcl-1 may counteract Bortezomib(VELCADE®) in CLL, AML and certain NHL cells. Consistent with this, thecytotoxic effects of proteasome inhibitors are enhanced when Mcl-1levels are contained at normal levels or reduced in a cell culture(Nencioni, et al. (2005) supra). This finding demonstrated that Mcl-1accumulation is an unwanted molecular consequence of exposure toproteasome inhibitors.

Examples of combination agents include, but are not limited to,alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone);a camptothecin (including the synthetic analogue topotecan); bryostatin;cally statin; CC-1065 (including its adozelesin, carzelesin andbizelesin synthetic analogues); cryptophycins (e.g., cryptophycin 1 andcryptophycin 8); dolastatin; duocarmycin (including the syntheticanalogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; asarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,chlornaphazine, cholophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gammall andcalicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, suchas clodronate; an esperamicin; as well as neocarzinostatin chromophoreand related chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinoymycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as minoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; def of amine;demecolcine; diaziquone; elformithine; elliptinium acetate; anepothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidainine; maytansinoids such as maytansine and ansamitocins;mitoguazone; mitoxantrone; mopidanmol; nitracrine; pentostatin;phenamet; pirarubicin; loxoxantrone; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlortriethylamine;trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine);urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipontomsn; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., TAXOL paclitaxel (Bristol-Myers SquibbOncology, Princeton, N.J.), ABRAXANE Cremophor-free, albumin-engineerednanoparticle formulation of paclitaxel (American PharmaceuticalPartners, Schaumberg, 111.), and TAXOTERE doxetaxel (Rhone-PoulencRorer, Antony, France); chloranbucil; GEMZAR gemcitabine; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin,oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16);ifosfamide; mitoxantrone; vincristine; NAVELBINE, vinorelbine;novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda;ibandronate; irinotecan (Camptosar, CPT-11) (including the treatmentregimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitorRFS 2000; difluormethylornithine (DMFO); retinoids such as retinoicacid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin,including the oxaliplatin treatment regimen (FOLFOX); lapatinib(Tykerb); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib(Tarceva)) and VEGF-A that reduce cell proliferation andpharmaceutically acceptable salts, acids or derivatives of any of theabove. In addition, the methods of treatment can further include the useof radiation.

C. Screening Methods

The present invention also teaches a method for selecting appropriateBH3 mimic compounds in treating particular tumors. This selection isbased on an understanding of the unique activity of compounds of FormulaI or II to mimic particular BH3 domains. Compounds from this group thathave unique activity against either all of the anti-apoptotic Bcl-2family proteins or a particular member of this family of proteins areuseful against particular tumors. Expression levels of particular Bcl-2family proteins can be assessed using standard assays, such as westernblot or immunohistological staining of biopsied tumor tissue. Followingthis assessment, compounds with activity against the elevated proteinsin the tumor sample will be selected as an appropriate therapeutic fortreating that tumor.

It is of particular interest to establish the correlation of Mcl-1expression levels to the occurrence of tumors. Based on the discoverythat compounds of Formula I or II inhibit Mcl-1 binding (see Example 1,below), it may be that cells that are hyper-dependent on Mcl-1, as aconsequence of elevated Mcl-1 in the disease state, will be sensitizedto compounds of Formula I or II or other derivative BH3 mimic compoundsthat are shown to inhibit Mcl-1.

BH3 mimic compounds that do inhibit Mcl-1 will become important secondline therapy for CLL given that tumor cells from patients who relapsefrom the current front line therapy Rituxan display high Mcl-1 to Baxratio (Bannerji, et al. (2003) J. Clin. Oncol. 21(8): 1466-71).Inhibition of Mcl-1 activity by a compound of Formula I or II, or otherBH3 mimic compounds will qualify those compounds as front linetherapeutics against MM, CLL, AML, ALL, and NHL as well as prostate,liver, and ovarian cancers and other malignancies that are affected byelevated Mcl-1.

There is a clear indication that elevated expression of anti-apoptoticBcl-2 family proteins is often the cause of chemoresistance(Kostanova-Poliakova, et al. (2005) Neoplasma 52(6): 441-9). Therefore,it will also be important to know which of the Bcl-2 family proteins areinvolved in chemoresistance when considering second line treatment forrelapsed patients.

The present invention also teaches a method for determining selectiveactivity of a BH3 mimic compound against the Bcl-2 family proteins. Thismethod also uses this information to predict efficacy of a selective BH3mimic compound in treating certain hematological malignancies that aredefined by expression levels of Bcl-2 family proteins. Specific activityagainst such disease-profiled Bcl-2 family proteins is predicted to havethe best anti-tumor activity and lowest toxicity against the non-tumorcells in the treated organism.

For instance, one skilled in the art can compare the blocking activityof BH3 mimic compounds to particular BH3 domain-containing peptides. Theactivity is the ability to block binding of certain BH3domain-containing Bcl-2 proteins. To do this, BH3 mimic compounds andparticular BH3-containing peptides are assessed for patterns of activityin inhibiting particular BH3 mediated protein/protein interactions.Patterns of activity are charted and an algorithm is used to determineoverlap between the activity of certain BH3 peptides and certain BH3mimic compounds. The ability to mimic particular BH3 domains isdetermined.

A compound that mimics a BH3 domain of a particular Bcl-2 family proteinwith a given physiological role will replicate the role of thatparticular BH3-containing protein. In a disease state where that proteinis known to have impaired function, the compound would replace thatparticular function.

The acquired understanding enables one skilled in the art to predict theefficacy of a given BH3 mimic compound in a given disease tissue type byrecognizing the protein with the most pronounced role in affecting thedisease state. This prediction is based on the understanding of the rolethat a sub-set of Bcl-2 family protein interactions or one particularprotein from this family plays in that tissue type and in the tissuespecific disease.

Recent work has described unique functions of BH3-only proteins inaffecting apoptosis. For instance, the BH3-only protein Bad, has beenidentified as having a “sensitizing” function compared to the BH3protein Bid which has an “effector” function in causing cell death(Letai, (2005) J. Clin. Invest. 115: 2648-2655). Additionally, BH3-onlyproteins have distinct roles in maintaining the healthy organism. Forinstance, the BH3-only protein Bim is required for thymocyte killing inresponse to negative selection signals. Bim function is essential forthe maintenance of the healthy thymocyte population. Bim recognizes cuessuch as cytoskeletal abnormalities while binding to themicrotubule-associated dynein motor complex (Puthalakath, et al. (1999)Mol Cell. 3(3): 287-96). The sensing mechanism and the role that thisBH3 protein plays are unique among all of the other BH3-only proteins.

BH3-only proteins have distinct roles in targeting particular members ofthe multi-domain Bcl-2 protein. The binding specificity of the variousBH3 domains to particular multi-domain Bcl-2 family proteins can affectdisease outcome. An example is the BH3-only protein Noxa that is highlyselective for binding to Mcl-1 and not other Bcl-2 family proteins(Chen, et al. (2005) Mol Cell. 17(3): 393-403). The pro-apoptoticprotein Bak is activated through Noxa-dependent displacement from Mcl-1(Willis, et al. (2005) Genes & Dev. 19(11): 1294-305). Noxa becomes amore significant death effector protein in the context of a high Mcl-1background, such as in lymphocytes and myeloid cells under certainconditions. Aberrant control of these interactions leads totyphomagenesis and myeloid cancer.

The BH3-only protein Puma is up-regulated by the oncogene p53 and isstrongly implicated in lymphomagenesis (Hemann, et al. (2004) Proc.Natl. Acad. Sci. USA 101: 9333-38). Deficiency in p53 activity causestumor formation. Expression of Puma in certain lymphoid cell backgroundsuppresses such p53 deficiency mediated tumorigenesis (Hemann, et al.(2004) Proc. Natl. Acad. Sci. USA 101: 9333-38).

The invention teaches that selection of a BH3 mimic compound thatmatches the binding inhibition activity pattern of the Noxa BH3 domainpeptide will result in a Noxa BH3 specific mimic. This compound willthen be useful in affecting diseases that involve the Noxa-Mcl-1interaction. These disease indications that would be served by such acompound/therapeutic include NHL, CLL, AML and ALL. A BH3 mimic compoundthat selectively mimics the Puma BH3 domain would suppress tumorsresulting from certain types of p53 deficiency by restoring deficientresponse to DNA damage-mediated apoptotic signals. A Bim BH3 mimiccompound is predicted to have selective efficacy against T-cells thathave lost responsiveness to negative selection as happens in certainautoimmune disease or thymocyte malignancies. Such a BH3 mimic wouldprovide an effective anti-autoimmune disease therapeutic for treatmentof diseases such as Type I diabetes, rheumatoid arthritis, multiplesclerosis, myasthenia gravis, psoriasis, lupus, inflammatory boweldisease, and other diseases.

Also contemplated is the exploitation of unique features of the BH3-onlyprotein Bad. Bad has been described as a sensitizing BH3 protein, unlikeBid or Bim that are effector BH3 proteins, capable of directlyactivating Bax or Bak and killing cells. It is therefore, likely thatmimetic compounds that selectively mimic Bad may have less toxicity thanthat which mimics the BH3 protein Bid and therefore will be well suitedas a general anti-tumor therapeutic.

The invention also teaches a method for recognizing small molecules thatfunction to mimic distinct members of the BH3 domain containing familyof proteins and in doing so, recognize small molecules more likely tobecome effective drug candidates.

D. Administration and Dosage

i. Routes of Administration

A compound of Formula I or II can be administered by any knownadministration method known to a person skilled in the art. Examples ofroutes of administration include but are not limited to oral,parenteral, intraperitoneal, intravenous, intraarterial, transdermal,topical, sublingual, intramuscular, rectal, transbuccal, intranasal,liposomal, via inhalation, vaginal, intraoccular, via local delivery bycatheter or stent, subcutaneous, intraadiposal, intraarticular,intrathecal, or in a controlled or extended release dosage form. Acompound of Formula I or II can be administered in accordance with anydose and dosing schedule that achieves a dose effective to treatdisease.

The route of administration of a compound of Formula I or II can beindependent of the route of administration of any additional anti-canceragents that are used. Either at least one of the compounds of Formula Ior II or another compound can be administered, for example, orally,parenterally, intraperitoneally, intravenously, intraarterially,transdermally, sublingually, intramuscularly, rectally, transbuccally,intranasally, liposomally, via inhalation, vaginally, intraoccularly,via local delivery by catheter or stent, subcutaneously,intraadiposally, intraarticularly, intrathecally, or in a controlled orextended release dosage form.

For example, a compound of the invention can be administered in oralforms, for example, as tablets, capsules (each of which includessustained release or timed release formulations), pills, powders,granules, elixirs, tinctures, suspensions, syrups, and emulsions.Likewise, a compound can be administered by intravenous (e.g., bolus orinfusion), intraperitoneal, subcutaneous, intramuscular, or other routesusing forms well known to those of ordinary skill in the pharmaceuticalarts. Particularly useful routes of administration of a compound areoral administration and intravenous delivery.

A compound can also be administered in the form of a depot injection orimplant preparation, which may be formulated in such a manner as topermit a sustained release of the active ingredient. The activeingredient can be compressed into pellets or small cylinders andimplanted subcutaneously or intramuscularly as depot injections orimplants. Implants may employ inert materials such as biodegradablepolymers or synthetic silicones, for example, Silastic, silicone rubberor other polymers manufactured by the Dow-Corning Corporation.

A compound can also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamellar vesiclesand multilamellar vesicles. Liposomes can be formed from a variety ofphospholipids, such as cholesterol, stearylamine, orphosphatidylcholines. Liposome versions of a compound may be used toincrease tolerance to the agents.

A compound can also be delivered by the use of monoclonal antibodies asindividual carriers to which the compound molecules are coupled.

A compound can also be prepared with soluble polymers as targetable drugcarriers. Such polymers can include polyvinylpyrrolidone, pyrancopolymer, polyhydroxy-propyl-methacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, a compound can beprepared with biodegradable polymers useful in achieving controlledrelease of a drug, for example, polylactic acid, polyglycolic acid,copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and cross linked or amphipathicblock copolymers of hydrogels.

In a specific embodiment, a compound can be administered orally in agelatin capsule, which can comprise excipients such as microcrystallinecellulose, croscarmellose sodium and magnesium stearate. For example, anembodiment can include 200 mg of solid compound with 89.5 mg ofmicrocrystalline cellulose, 9 mg of sodium croscarmellose, and 1.5 mg ofmagnesium stearate contained in a gelatin capsule.

ii. Dosages and Dosage Schedules

The dosage regimen utilizing a compound of Formula I or II can beselected in accordance with a variety of factors including type,species, age, weight, and sex of the patient; the type of disease beingtreated; the severity (e.g., stage) of the disease to be treated; theroute of administration; the renal and hepatic function of the patient;and the particular compound or salt thereof employed, among others. Adosage regimen can be used, for example, to prevent, inhibit (fully orpartially), or arrest the progress of the disease.

In accordance with the invention, a compound of Formula I or II can beadministered by continuous or intermittent dosages. For example,intermittent administration of a compound of Formula I or II may beadministered one to six days per week or it may be administered incycles with rest periods in between the cycles (e.g., dailyadministration for two to eight consecutive weeks, then a rest periodwith no administration for up to one week between treatments) or it maybe administered on alternate days.

For example, in one embodiment, a compound of Formula I or II can beadministered in a total daily dose of up to 800 mg. A compound ofFormula I or II can be administered once daily (QD), or divided intomultiple daily doses such as twice daily (BID), and three times daily(YID). A compound of Formula I or II, can be administered at a totaldaily dosage of up to 800 mg, for example, about 200 mg, 300 mg, 400 mg,600 mg, or 800 mg, which can be administered in one daily dose or can bedivided into multiple daily doses as described above. In specificaspects, the administration is oral or by intravenous delivery.

In one embodiment, the compound is administered once daily at a dose ofabout 200-600 mg. In another embodiment, the compound is administeredtwice daily at a dose of about 200-400 mg. In another embodiment, thecompound is administered twice daily at a dose of about 200-400 mgintermittently, for example three, four or five days per week. In oneembodiment, the daily dose is about 200 mg which can be administeredonce-daily, twice-daily or three-times daily. In one embodiment, thedaily dose is about 300 mg which can be administered once-daily,twice-daily or three-times daily. In one embodiment, the daily dose isabout 400 mg which can be administered once-daily, twice-daily orthree-times daily.

A compound of Formula I or II, can be administered in accordance withany dose and dosing schedule that achieves a dose effective to treatcancer. Each compound can be administered in a total daily dose that mayvary from patient to patient, and may be administered at varying dosageschedules. For example, a compound of the invention can be administeredto the patient at a total daily dosage of between 25-4000 mg/m². Inparticular, a compound of Formula I or II can be administered in a totaldaily dose of up to 800 mg, including by oral or intravenousadministration, once, twice or three times daily, continuously (everyday) or intermittently (e.g., 3-5 days a week). In addition, theadministration can be continuous, e.g., every day, or intermittently.

In addition, a compound of Formula I or II may be administered accordingto any of the schedules described above, consecutively for a few weeks,followed by a rest period.

In one embodiment, the patient can receive intravenously orsubcutaneously compounds of Formula I or II in quantities sufficient todeliver between about 3-1500 mg/m² per day, for example, about 3, 30,60, 90, 180, 300, 600, 900, 1200 or 1500 mg/m² per day. Such quantitiesmay be administered in a number of suitable ways, e.g. large volumes oflow concentrations of the compounds of Formula I or II can be usedduring one extended period of time or several times a day. Thequantities can be administered for one or more consecutive days,intermittent days or a combination thereof per week (7 day period).Alternatively, low volumes of high concentrations of the compounds ofFormula I or II can be used during a short period of time, e.g. once aday for one or more days either consecutively, intermittently or acombination thereof per week (7 day period). For example, a dose of 300mg/m² per day can be administered for 5 consecutive days for a total ofabout 1500 mg/m² per treatment. In another dosing regimen, the number ofconsecutive days can also be 5, with treatment lasting for 2 or 3consecutive weeks for a total of about 3000 mg/m² or about 4500 mg/m²total treatment.

Typically, an intravenous formulation may be prepared which contains aconcentration of a compound of Formula I or II of between about 1.0mg/mL to about 10 mg/mL, e.g. about 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, 5.0mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL and 10 mg/mL andadministered in amounts to achieve the doses described above. In oneexample, a sufficient volume of intravenous formulation can beadministered to a patient in a day such that the total dose for the dayis between about 300 and about 1500 mg/m².

Subcutaneous formulations can be prepared according to procedures wellknown in the art at a pH in the range between about 5 and about 12,which include suitable buffers and are tonicity agents, as describedbelow. They can be formulated to deliver a daily dose of any ofcompounds of Formula I or II in one or more daily subcutaneousadministrations, for example, one, two or three times each day.

It is apparent to a person skilled in the art that any one or more ofthe specific dosages and dosage schedules of a compound of Formula I orII are also applicable to any one or more of the anti-cancer agents tobe used in a combination treatment. Moreover, the specific dosage anddosage schedule of a compound of Formula I or II can further vary, andthe optimal dose, dosing schedule, and route of administration can bedetermined based upon the specific drug combination that is being used.Further, the various modes of administration, dosages, and dosingschedules described herein merely set forth specific embodiments andshould not be construed as limiting the broad scope of the invention.Any permutations, variations, and combinations of the dosages and dosingschedules are included within the scope of the present invention.

iii. Formulation

An “effective amount” of a compound of Formula I or II is the quantitywhich, when administered to a subject having a disease or disorder,results in regression of the disease or disorder in the subject. Thus,an effective amount of a compound of the disclosed invention is thequantity which, when administered to a subject having a cellproliferation disorder, results in, for example, regression of cellgrowth or cell death in a subject. The amount of the disclosed compoundto be administered to a subject will depend on the particular disorder,the mode of administration, co-administered compounds, if any, and thecharacteristics of the subject, such as general health, other diseases,age, sex, genotype, body weight and tolerance to drugs. The skilledartisan will be able to determine appropriate dosages depending on theseand other factors.

As used herein, the term “effective amount” refers to an amount of acompound, or a combination of compounds, of the present inventioneffective when administered alone or in combination as ananti-proliferative agent. For example, an effective amount refers to anamount of the compound present in a formulation or on a medical devicegiven to a recipient patient or subject sufficient to elicit biologicalactivity, for example, anti-proliferative activity, such as for example,anti-cancer activity or anti-neoplastic activity. The combination ofcompounds optionally is a synergistic combination. Synergy, asdescribed, for example, by Chou and Talalay, (1984) Adv. Enzyme Regul.22: 27-55, occurs when the effect of the compounds when administered incombination is greater than the additive effect of the compounds whenadministered alone as a single agent. In general, a synergistic effectis most clearly demonstrated at sub-optimal concentrations of thecompounds. Synergy can be in terms of lower cytotoxicity, or increasedanti-proliferative effect, or some other beneficial effect of thecombination compared with the individual components.

A “therapeutically effective amount” means the amount of a compoundthat, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the mammal tobe treated.

A therapeutically effective amount of one or more of a compound can beformulated with a pharmaceutically acceptable carrier for administrationto a human or an animal. Accordingly, a compound or a formulation can beadministered, for example, via oral, parenteral, or topical routes, toprovide an effective amount of the compound. In alternative embodiments,a compound is prepared in accordance with the present invention can beused to coat or impregnate a medical device, e.g., a stent.

The term “prophylactically effective amount” means an effective amountof a compound or compounds, of the present invention that isadministered to prevent or reduce the risk of unwanted cellularproliferation.

“Pharmacological effect” as used herein encompasses effects produced inthe subject that achieve the intended purpose of a therapy. In oneembodiment, a pharmacological effect means that primary indications ofthe subject being treated are prevented, alleviated, or reduced. Forexample, a pharmacological effect would be one that results in theprevention, alleviation or reduction of primary indications in a treatedsubject. In another embodiment, a pharmacological effect means thatdisorders or symptoms of the primary indications of the subject beingtreated are prevented, alleviated, or reduced. For example, apharmacological effect would be one that results in the prevention orreduction of primary indications in a treated subject.

A “pharmaceutical composition” is a formulation containing a compound ofFormula I or II in a form suitable for administration to a subject. Inone embodiment, the pharmaceutical composition is in bulk or in unitdosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler, or a vial. The quantity of active ingredient (e.g.,a formulation of the compound or salt, hydrate, solvate, or isomerthereof) in a unit dose of composition is an effective amount and isvaried according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration, Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” caninclude both one and more than one such excipient.

A compound of Formula I or II is capable of further forming salts. Allof these forms are also contemplated within the scope of the claimedinvention.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. As used herein,“pharmaceutically acceptable salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines, alkali or organic salts of acidicresidues such as carboxylic acids, and the like. The pharmaceuticallyacceptable salts include the conventional non-toxic salts or thequaternary ammonium salts of the parent compound formed, for example,from non-toxic inorganic or organic acids. For example, suchconventional non-toxic salts include, but are not limited to, thosederived from inorganic and organic acids selected from 2-acetoxybenzoic,2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic,bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic,glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic,hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic isethionic,lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methanesulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic,phosphoric, polygalacturonic, propionic, salicyclic, stearic, subacetic,succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluenesulfonic, and the commonly occurring amine acids, e.g., glycine,alanine, phenylalanine, arginine, etc.

Other examples include hexanoic acid, cyclopentane propionic acid,pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamicacid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, andthe like. The invention also encompasses salts formed when an acidicproton present in the parent compound either is replaced by a metal ion,e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; orcoordinates with an organic base such as ethanolamine, diethanolamine,triethanolamine, tromethamine, N-methyglucamine, and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The pharmaceutically acceptable salts of a compound of Formula I or IIcan be synthesized from a parent compound that contains a basic oracidic moiety by conventional chemical methods. Generally, such saltscan be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, non-aqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile may be used. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 18th ed. (Mack PublishingCompany, 1990). For example, salts can include, but are not limited to,the hydrochloride and acetate salts of the aliphatic amine-containing,hydroxyl amine-containing and imine-containing compounds of the presentinvention.

A compound of Formula I or II can also be prepared as esters, forexample pharmaceutically acceptable esters. For example a carboxylicacid functional group in a compound can be converted to itscorresponding ester, e.g., a methyl, ethyl, or other ester. Also, analcohol group in a compound can be converted to its corresponding ester,e.g., an acetate, propionate, or other ester.

A compound of Formula I or II can also be prepared as prodrugs, forexample pharmaceutically acceptable prodrugs. The terms “pro-drug” and“prodrug” are used interchangeably herein and refer to any compound thatreleases an active parent drug in vivo. Since prodrugs are known toenhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.) a compound of thepresent invention can be delivered in prodrug form. Thus, the presentinvention is intended to cover prodrugs of the presently claimedcompounds, methods of delivering the same and compositions containingthe same. “Prodrugs” are intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when such prodrug is administered to a subject. Prodrugs of thepresent invention are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds of the present invention wherein a hydroxyl, amino,sulfhydryl, carboxyl, or carbonyl group is bonded to any group that maybe cleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl,free carboxyl or free carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates, andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxyl functional groups, ester groups (e.g. ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g. N-acetyl), N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds of theinvention including compounds of Formula I or II or derivatives, and thelike, (see, Bundegaard, “Design of Prodrugs” pgs. 1-92, Elsevier, NewYork-Oxford (1985)).

All percentages and ratios used herein, unless otherwise indicated, areby weight.

“Combination therapy” (or “co-therapy”) includes the administration of acompound of Formula I or II and at least a second agent as part of aspecific treatment regimen intended to provide the beneficial effectfrom the co-action of these therapeutic agents. The beneficial effect ofthe combination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time period (usually minutes,hours, days or weeks, depending upon the combination selected).“Combination therapy” may, but generally is not, intended to encompassthe administration of two or more of these therapeutic agents as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present invention.

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anyappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. The therapeutic agents can be administered bythe same route or by different routes. For example, a first therapeuticagent of the combination selected may be administered by intravenousinjection while the other therapeutic agents of the combination may beadministered orally. Alternatively, for example, all therapeutic agentsmay be administered orally or all therapeutic agents may be administeredby intravenous injection. The sequence in which the therapeutic agentsare administered is not narrowly critical.

“Combination therapy” also embraces the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery orradiation treatment). Where the combination therapy further comprises anon-drug treatment, the non-drug treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where processes are described as having,including, or comprising specific process steps, the processes alsoconsist essentially of, or consist of, the recited processing steps.Further, it should be understood that the order of steps or order forperforming certain actions are immaterial so long as the inventionremains operable. Moreover, two or more steps or actions may beconducted simultaneously.

A compound of Formula I or II, or pharmaceutically acceptable saltsthereof, can be administered orally, nasally, transdermally, pulmonary,inhalationally, buccally, sublingually, intraperintoneally,subcutaneously, intramuscularly, intravenously, rectally,intrapleurally, intrathecally and parenterally. In certain embodiments,the compound is administered orally. One skilled in the art willrecognize the advantages of certain routes of administration.

The dosage regimen utilizing the a compound is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) ed., Mack Publishing Co., Easton, Pa. (1995). In anembodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

In one embodiment, a compound of Formula I or II are prepared for oraladministration, wherein the disclosed compounds or salts thereof arecombined with a suitable solid or liquid carrier or diluent to formcapsules, tablets, pills, powders, syrups, solutions, suspensions andthe like.

The tablets, pills, capsules, and the like contain from about 1 to about99 weight percent of the active ingredient and a binder such as gumtragacanth, acacias, corn starch or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch or alginic acid; a lubricant such as magnesium stearate; and/or asweetening agent such as sucrose, lactose, saccharin, xylitol, and thelike. When a dosage unit form is a capsule, it often contains, inaddition to materials of the above type, a liquid carrier such as fattyoil.

In some embodiments, various other materials are present as coatings orto modify the physical form of the dosage unit. For instance, in someembodiments, tablets are coated with shellac, sugar or both. In someembodiments, a syrup or elixir contains, in addition to the activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and a flavoring such as cherry or orange flavor,and the like.

For some embodiments relating to parental administration, a compound ofFormula I or II or salts, solvates, tautomers or polymorphs thereof, canbe combined with sterile aqueous or organic media to form injectablesolutions or suspensions. Injectable compositions may be aqueousisotonic solutions or suspensions. The compositions may be sterilizedand/or contain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and contain about 0.1 to 75%, or about 1 to 50%, of theactive ingredient.

For example, injectable solutions are produced using solvents such assesame or peanut oil or aqueous propylene glycol, as well as aqueoussolutions of water-soluble pharmaceutically-acceptable salts of thecompounds. In some embodiments, dispersions are prepared in glycerol,liquid polyethylene glycols and mixtures thereof in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. The terms “parenteraladministration” and “administered parenterally” as used herein meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal andintrasternal injection and infusion.

For rectal administration, suitable pharmaceutical compositions are, forexample, topical preparations, suppositories or enemas. Suppositoriesare advantageously prepared from fatty emulsions or suspensions. Thecompositions may be sterilized and/or contain adjuvants, such aspreserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating the osmotic pressure and/or buffers. Inaddition, they may also contain other therapeutically valuablesubstances. The compositions are prepared according to conventionalmixing, granulating or coating methods, respectively, and contain about0.1 to 75%, or about 1 to 50%, of the active ingredient.

In some embodiments, a compound of Formula I or II is formulated todeliver the active agent by pulmonary administration, e.g.,administration of an aerosol formulation containing the active agentfrom, for example, a manual pump spray, nebulizer or pressurizedmetered-dose inhaler. In some embodiments, suitable formulations of thistype also include other agents, such as antistatic agents, to maintainthe disclosed compounds as effective aerosols.

A drug delivery device for delivering aerosols comprises a suitableaerosol canister with a metering valve containing a pharmaceuticalaerosol formulation as described and an actuator housing adapted to holdthe canister and allow for drug delivery. The canister in the drugdelivery device has a headspace representing greater than about 15% ofthe total volume of the canister. Often, the polymer intended forpulmonary administration is dissolved, suspended or emulsified in amixture of a solvent, surfactant and propellant. The mixture ismaintained under pressure in a canister that has been sealed with ametering valve.

For nasal administration, either a solid or a liquid carrier can beused. The solid carrier includes a coarse powder having particle size inthe range of, for example, from about 20 to about 500 microns and suchformulation is administered by inhalation through the nasal passages. Insome embodiments where the liquid carrier is used, the formulation isadministered as a nasal spray or drops and includes oil or aqueoussolutions of the active ingredients.

Also contemplated are formulations that are rapidly dispersing dosageforms, also known as “flash dose” forms. In particular, some embodimentsof the present invention are formulated as compositions that releasetheir active ingredients within a short period of time, for example,typically less than about five minutes, less than about ninety seconds,less than about thirty seconds and less than about ten or fifteenseconds. Such formulations are suitable for administration to a subjectvia a variety of routes, for example by insertion into a body cavity orapplication to a moist body surface or open wound.

Typically, a “flash dosage” is a solid dosage form that is administeredorally, which rapidly disperses in the mouth, and hence does not requiregreat effort in swallowing and allows the compound to be rapidlyingested or absorbed through the oral mucosal membranes. In someembodiments, suitable rapidly dispersing dosage forms are also used inother applications, including the treatment of wounds and other bodilyinsults and diseased states in which release of the medicament byexternally supplied moisture is not possible.

“Flash dose” forms are known in the art; see for example, effervescentdosage forms and quick release coatings of insoluble microparticles inU.S. Pat. Nos. 5,578,322 and 5,607,697; freeze dried foams and liquidsin U.S. Pat. Nos. 4,642,903 and 5,631,023; melt spinning of dosage formsin U.S. Pat. Nos. 4,855,326, 5,380,473 and 5,518,730; solid, free-formfabrication in U.S. Pat. No. 6,471,992; saccharide-based carrier matrixand a liquid binder in U.S. Pat. Nos. 5,587,172, 5,616,344, 6,277,406,and 5,622,719; and other forms known to the art.

A compound of Formula I or II can also be also formulated as “pulsedrelease” formulations, in which the compound is released from thepharmaceutical compositions in a series of releases (i.e., pulses). Thecompounds are also formulated as “sustained release” formulations inwhich the compound is continuously released from the pharmaceuticalcomposition over a prolonged period.

Also contemplated are formulations, for example, liquid formulations,including cyclic or acyclic encapsulating or solvating agents, forexample, cyclodextrins, polyethers, or polysaccharides (e.g.,methylcellulose), or polyanionic α-cyclodextrin derivatives with asodium sulfonate salt group separate from the lipophilic cavity by analkyl ether spacer group or polysaccharides. In one embodiment, theagent can be polyanionic cyclodextrin derivative with a sodium sulfonatesalt separated from the lipophilic cavity by a butyl ether spacer group,e.g., CAPTISOL® (CyDex, Overland, and KS). One skilled in the art canevaluate suitable agent/disclosed compound formulation ratios bypreparing a solution of the agent in water, e.g., a 40% by weightsolution; preparing serial dilutions, e.g. to make solutions of 20%, 10,5%, 2.5%, 0% (control), and the like; adding an excess (compared to theamount that can be solubilized by the agent) of the disclosed compound;mixing under appropriate conditions, e.g., heating, agitation,sonication, and the like; centrifuging or filtering the resultingmixtures to obtain clear solutions; and analyzing the solutions forconcentration of the disclosed compound.

The invention is illustrated in the examples that follow. This sectionis set forth to aid in an understanding of the invention but is notintended to, and should not be construed to limit in any way theinvention as set forth in the claims which follow thereafter.

Methods of Making Preparation of6-((4-ethylpiperazin-1-yl)(4-(trifluoromethyl)phenyl)methyl)isoquinolin-5-ol(1)

A mixture of isoquinolin-5-ol (1 eq), 4-(trifluoromethyl)benzaldehyde (1eq) and 1-ethylpiperazine (1 eq) was heated overnight at 110° C. in anoil bath. The solution was cooled to room temperature and the mixturewas precipitated in ethanol. The solid was collected by filtration toyield the title compound (12.6 mg, yellow solid) in high purity (>95%)as judged by ¹H NMR and LCMS analysis. MS (M+1) cal'd=416.2, found416.2. ¹H NMR (400 MHz, DMSO-d₆) δ 13.12 (br s, 1H), 9.08 (s, 1H), 8.54(d, 1H), 7.92 (d, 1H), 7.69 (d, 2H), 7.64 (d, 2H), 7.41 (d, 1H), 7.19(d, 1H), 4.75 (s, 1H), 2.45-2.55 (broad m, 8H), 2.35 (q, 2H), 1.02 (t,3H).

Preparation of7-((4-ethylpiperazin-1-yl)(4-(trifluoromethyl)phenyl)methyl)-8-methoxyquinoline(8)

A mixture of quinolin-8-ol (1 eq), 4-fluorobenzaldehyde (1 eq) and1-ethylpiperazine (1 eq) was heated overnight at 110° C. in an oil bath.The solution was cooled to room temperature and the mixture wasprecipitated in ethanol. The solid was collected by filtration to yieldthe crude intermediate7-((4-ethylpiperazin-1-yl)(4-(trifluoromethyl)phenyl)methyl)quinolin-8-ol.

A mixture of7-((4-ethylpiperazin-1-yl)(4-(trifluoromethyl)phenyl)methyl)quinolin-8-ol(1 eq) in diethyl ether was prepared and cooled to 0° C. To this wasadded a solution of diazomethane in diethyl ether (excess) and thesolution was stirred at this temperature for 1 hour. Following standardaqueous workup, the solid was collected by filtration to provide7-((4-ethylpiperazin-1-yl)(4-(trifluoromethyl)phenyl)methyl)-8-methoxyquinoline(5.0 mgs, 8% yield) in high purity (>95%) as judged by ¹H NMR and LCMSanalysis. MS (M+1) cal'd=430.5, found 430.4. ¹H NMR (400 MHz, DMSO-d₆) δ8.88 (dd, 1H), 8.30 (dd, 1H), 7.77 (d, 1H), 7.67 (m, 5H), 7.52 (m, 1H),5.10 (s, 1H), 4.12 (s, 3H), 2.35 (m, 2H), 2.25-2.40 (broad m, 8H), 0.97(m, 3H).

EXAMPLES

The disclosure is further illustrated by the following examples, whichare not to be construed as limiting this disclosure in scope or spiritto the specific procedures herein described. It is to be understood thatthe examples are provided to illustrate certain embodiments and that nolimitation to the scope of the disclosure is intended thereby. It is tobe further understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which may suggestthemselves to those skilled in the art without departing from the spiritof the present disclosure and/or scope of the appended claims.

Example 1. Inhibition of Mcl-1 by Compounds of Formula I or Formula II;Measurement by ELISA Assay

The expression level of Mcl-1 correlates directly to chemo-sensitivityand survival of certain non-Hodgkin's lymphomas (Petlickovsk, et al.(2005) Blood 105(12): 4820-7) as well as prostate cancer (Royuela, etal. (2001) Eur. Cytokine Netw. 12(4): 654-63), liver cancer (Fleischer,et al. (2006) Int. J. Oncol. 28(1): 25-32) and other cancers. Mcl-1 istherefore an ideal target for treating these cancers. This example showsthat the BH3 mimic compounds of Formula I or Formula II inhibit thebinding of the BH3 domain of the Bcl-2 family protein Bim to Mcl-1.Accordingly, this example indicates that compounds of Formula I orFormula II are effective in treating certain hematological malignanciesthat are affected principally by the Bcl-2 family protein Mcl-1.

Materials and Methods

An ELISA-like streptavidin plate assay was used to demonstrate theactivity of the BH3 mimic compounds of Formula I or Formula II toinhibit Mcl-1 to Bim BH3 as described in Wang, et al. (2006) J Med Chem49: 6139-6142.

Recombinant GST-Mcl-1 fusion protein, used as described below, wasgenerated in E. coli and purified using glutathione-sepharose beadsusing conventional techniques known to those skilled in the art (methodsfor preparation are described in, e.g., Strategies for ProteinPurification and Characterization, Marshak, et al. CSH press, ColdSpring Harbor, N.Y.). Binding of the recombinant proteins to thefluorescent Bim BH3 domain was confirmed by titration of increasingconcentrations of the recombinant proteins against a constant amount oflabeled peptide (4 nM). Quantitation of binding was accomplished by FPassay with mP measurements made on the Analyst-GT reader (MolecularDevices, Sunnyvale, Calif.).

A streptavidin-coated plate (Thermo Scientific, NUNC #436014) was washedthree times with 300 μL of PBS-0.05% Tween solution. A biotinylatedtwenty-six amino acid peptide, corresponding to the BH3 domain of Bim,with the sequencebiotin-(β)A-D-M-R-P-E-I-W-I-A-Q-E-L-R-R-I-G-D-E-F-N-A-Y-Y-A-R-R-amide(SEQ ID No. 14), hereafter referred to as biotin-Bim, was obtained(Tufts). The biotin-Birn peptide was diluted to 0.018 μg/mL (5 nM) inSuperBlock blocking buffer in PBS (Thermo Scientific, #37515), and 100μL of this solution was incubated in the streptavidin-coated plate for2.5 hours while shaking. Separately, a compound of Formula I or FormulaII, which had been prepared in a 10 mM stock DMSO solution, was thenincubated with 20 nM GST-Mcl-1 fusion protein in PBS. Compounds weretested in six three-fold dilutions ranging from 20 μM to 0.6 μM, 10 μMto 0.3 μM, or 5 μM to 0.15 μM. n addition, Bim peptide (New EnglandPeptide) was utilized as a control and tested in six-fold dilutionsranging from 100 nM to 3.3 nM. The streptavidin-coated plate which hadpreviously been treated with biotin-Bim peptide was then washed threetimes with 300 μL of PBS-0.05% Tween solution. A solution of GST-MCL-1and a compound of Formula I or Formula II (100 uL) was then added to thestreptavidin-coated plate, with two wells utilized as controls (PBSbuffer only, no GST-MCL-1 or biotin-Bim) and two wells as an alternativecontrol (containing GST-MCL-1 but no biotin-Bim), and four three-folddilutions of DMSO (0.2%, 0.06%, 0.02%, and 0.008%). All wells containingcompounds of Formula I or Formula II were loaded in duplicate. The platewas incubated for 2 hours at RT, then washed three times with 300 μL ofPBS-0.05% Tween solution. Anti-GST HRP (GE Healthcare, #RPN1236) isdiluted 1:20,000 in freshly prepared PBS, 0.1% Tween20, and 0.5% BSA wasthen added to the plate and the plate was incubated for 30 minutes. Theplate was then washed five times with 300 μL of PBS-0.05% Tweensolution. A solution of color reagents A (stabilized peroxide solution)and B (stabilized chrommogen solution) (R&D Systems, #DY999) is mixed ina 1:1 ratio and added to each well in a quantity of 80 uL. The wellswere allowed to develop until control wells containing DMSO are blue(approximately 5-10 minutes). A solution of ELISA stop solution (1Msulfuric acid, 20 uL per well) was then added. Absorbance at 450 nM wasread on a Tecan GeniosPro plate reader. Percent inhibition was thencalculated as follows: Percent inhibition=1−((Absorbance−Absorbance ofPBS-only well)/(Average Absorbance DMSO well−Absorbance of PBS-onlywell))×100.

In the calculation above, the average absorbance calculated for eachcompound of Formula I or Formula II (of two wells) was calculated andutilized to calculate the percent inhibition. Percent inhibition wasthen utilized to calculate IC50 values for each compound of Formula I orFormula II.

Results

Compounds of Formula I or Formula II were effective at inhibiting theBim-BH3 peptide front Mcl-1 with a drug concentration that provokes aresponse halfway between baseline and maximum (IC₅₀) that ranged from0.4 μM-16.7 μM, as shown in FIG. 1.

Example 2. Inhibition of Mcl-1 and Bcl-xL by Compounds of Formula I orII; Measurement by Fluorescence Polarization Assay

In this example, a Fluorescence Polarization (FP) assay is used todemonstrate the activity of the Mcl-1 inhibitors described in Formulas Ior II in inhibiting Mcl-1 as well as Bcl-xL binding to Bim BH3 asdescribed in Degterev et al. (2001) Nature Cell Biology 3: 173-182.

A nineteen amino acid peptide, corresponding to the BH3 domain of Bim,with the sequence FITC-GGGIAQELRRIGDEFNAY (SEQ ID NO: 1) is labeled withthe fluorophore FITC according to the manufacturer's instructions(Molecular Probes, Eugene, Oreg.). This sequence is identified as beingable to bind to purified Bcl-xL protein (Sather et al. (1997) Science275(5302): 983-86) and to have biological activity in cells (Holinger etal. J. Biol. Chem. 274: 13298-1330).

In addition, recombinant GST-Mcl-1 and GST-Bcl-xL fusion proteins aregenerated in E. coli and purified using glutathione-sepharose beadsusing conventional techniques known to those skilled in the art (methodsfor preparation are described in Strategies for Protein Purification andCharacterization, Marshak, D, Kadonga, J, Burgess R, Knuth, M, Brennan,W, Sue-Hwa, L, CSH press, Cold Spring Harbor, N.Y.). Binding of therecombinant proteins to the fluorescent Bim BH3 domain is confirmed bytitration of increasing concentrations of the recombinant proteinsagainst a constant amount of labeled Bim peptide (16.65 nM).Quantitation of binding is accomplished by FP assay with mP measurementsmade on the Analyst-GT reader (Molecular Devices, Sunnyvale, Calif.)

Further, the ability of the compounds of this invention as described inFormulas I or II to disrupt the interaction between the fluorescent BimBH3 peptide and the two fusion proteins is assessed using a fluorescencepolarization assay. The Bim peptide (in solution at 4 nano-Molar) andeither GST-Mcl-1 (in solution at 12.5 nM) or GST-Bcl-xL (in solution at11.8 nM) are first combined together in phosphate buffered salinebuffer, and then the compound solution in DMSO is added to finalconcentrations ranging from 10 μM to 0.1 nM), Normally the unbound Bimpolypeptide results in polarization of 5 mP units. Upon binding of theGST-Bcl-xL and GST-Mcl-1 fusion protein, polarization increases to 100mP units.

Results

Compounds of Formula I or II are effective at inhibiting Mcl-1 as wellas Bcl-xL binding to Bim BH3.

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in any manner. The content of anyindividual section may be equally applicable to all sections.

What is claimed is:
 1. A method for inhibiting Bcl-2 family proteinMcl-1 from binding to Bim BH3, comprising administering to a patient aneffective amount of a compound of Formula I or Formula II:

or a stereoisomer thereof, tautomer thereof, or a pharmaceuticallyacceptable salt thereof, wherein: X is O, C, or N, with C or Nsubstituted with hydrogen, C₁₋₆ alkyl, hydroxyl-substituted alkyl, C₅₋₁₀aryl, or a carbonyl group; wherein said alkyl, hydroxyl-substitutedalkyl, or aryl group is substituted with one or more C₁-C₆ alkyl, C₃-C₁₀cycloalkyl, C₁-C₆ perfluoroalkyl, halo, haloalkyl, hydroxyl, C₁-C₆hydroxylalkyl, —NH₂, aminoalkyl, dialkylamino, —COOH, carboxylic ester,primary, secondary, or tertiary carboxylic amide, urea, —C(O)O—(C₁-C₆alkyl), —OC(O)(C₁-C₆ alkyl), N-alkylamido, —C(O)NH₂, (C₁-C₆ alkyl)amido,—S(O)₂N-alkyl, —S(O)₂N-aryl, alkoxy, and —NO₂; Y is CH or N; Z is C orN; Z′ is CH or N; when Z is C, R₁ is hydrogen, alkoxy, perfluoroalkyl,F, Cl; and when Z is N, R₁ is null; R₃ is selected from C₁₋₆perfluoroalkyl, straight or branched C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ cycloalkyl, C₅₋₈ cycloalkenyl, C₁₋₁₀ alkylamino; whereinsaid perfluoroalkyl, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,or alkylamino group is substituted with one or more C₁-C₆ alkyl, C₃-C₁₀cycloalkyl, C₁-C₆ perfluoroalkyl, halo, haloalkyl, hydroxyl, C₁-C₆hydroxylalkyl, —NH₂, aminoalkyl, dialkylamino, —COOH, carboxylic ester,primary, secondary, or tertiary carboxylic amide, urea, —C(O)O—(C₁-C₆alkyl), —OC(O)(C₁-C₆ alkyl), N-alkylamido, —C(O)NH₂, (C₁-C₆ alkyl)amido,—S(O)₂N-alkyl, —S(O)₂N-aryl, alkoxy, and —NO₂; and R₄ is hydrogen,halogen, C₁₋₆ perfluoroalkyl, straight or branched C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ cycloalkyl, C₅₋₈ cycloalkenyl, C₁₋₁₀alkylamino, or C₅₋₁₀ aryl, or saturated or unsaturated 3-11 memberheteroaryl or heteroarylalkyl consisting of 1, 2, 3, and 4 heteroatomsselected independently from N, O, S, and S(O)₂, wherein saidperfluoroalkyl, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,alkylamino, aryl, heteroaryl or heteroarylalkyl group is substitutedwith one or more C₁-C₆ alkyl, C₃-C₁₀ cycloalkyl, C₁-C₆ perfluoroalkyl,halo, haloalkyl, hydroxyl, C₁-C₆ hydroxylalkyl, —NH₂, aminoalkyl,dialkylamino, —COOH, carboxylic ester, primary, secondary, or tertiarycarboxylic amide, urea, —C(O)O—(C₁-C₆ alkyl), —OC(O)(C₁-C₆ alkyl),N-alkylamido, —C(O)NH₂, (C₁-C₆ alkyl)amido, —S(O)₂N-alkyl, —S(O)₂N-aryl,alkoxy, and —NO₂, wherein the compound of Formula I or Formula IIinhibits the activity of the Bcl-2 family protein Mcl-1 from binding toBim BH3.
 2. The method of claim 1, wherein the compound of Formula I orFormula II is selected from the group consisting of:6-((4-ethylpiperazin-1-yl)(4-(trifluoromethyl)phenyl)methyl)isoquinolin-5-ol(1); tert-butyl4-((4-fluorophenyl)(5-hydroxyisoquinolin-6-yl)methyl)piperazine-1-carboxylate(2); 6-((4-ethylpiperazin-1-yl)(4-methoxyphenyl)methyl)isoquinolin-5-ol(3); 6-((4-ethylpiperazin-1-yl)(4-fluorophenyl)methyl)isoquinolin-5-ol(4); 6-(morpholino(4-(trifluoromethyl)phenyl)methyl)isoquinolin-5-ol(5); 6-((4-fluorophenyl)(morpholino)methyl)isoquinolin-5-ol (6)6-((4-methoxyphenyl)(morpholino)methyl)isoquinolin-5-ol (7);7-((4-ethylpiperazin-1-yl)(4-(trifluoromethyl)phenyl)methyl)-8-methoxyquinoline(8); 7-((4ethylpiperazin-1-yl)(4-fluorophenyl)methyl)-8-methoxyquinoline(9); and 4-((4-fluorophenyl)(8-methoxyquinolin-7-yl)methyl)morpholine(10).
 3. The method of claim 1, wherein the patient is also administereda 26S proteasome inhibitor.
 4. The method of claim 3, wherein the 26Sproteasome inhibitor is bortezomib.
 5. The method of claim 1, whereinthe patient is also administered dexamethasone, lenolidomide,thalidomide, or a combination thereof.
 6. The method of claim 1, whereinthe patient is also administered a chemotherapeutic agent that increasesthe level of Mcl-1.
 7. The method of claim 1, wherein the compound ofFormula I is6-((4-ethylpiperazin-1-yl)(4-(trifluoromethyl)phenyl)methyl)isoquinolin-5-ol(1).